Single nucleotide polymorphisms and mutations on Alpha-2-Macroglobulin

ABSTRACT

The present invention is related to the discovery of several single nucleotide polymorphisms (SNPs) and/or mutations in the Alpha-2-Macroglobulin gene (A2M), which are risk factors for Alzheimer&#39;s Disease (AD). More specifically, aspects of the invention concern nucleic acids corresponding to the A2M gene or fragments thereof, which contain one or more of the SNPs and/or mutations described herein, peptides or proteins encoded by said nucleic acids, antibodies to said peptides or proteins and methods of making said compositions, diagnostic methods, methods of data analysis, and pharmaceutical discovery and preparation methods.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional PatentApplication No. 60/337,434, entitled SINGLE NUCLEOTIDE POLYMORPHISMS ANDMUTATIONS ON ALPHA-2 MACROGLOBULIN, filed Nov. 9, 2001, the disclosureof which is incorporated herein by reference in its entirety. Thisapplication is also related to the Patent Cooperation Treaty Applicationhaving the Attorney Docket Number NEURINC.009VPC, entitled SINGLENUCLEOTIDE POLYMORPHISMS AND MUTATIONS ON ALPHA-2-MACROGLOBULIN, filedon Nov. 8, 2002, the disclosure of which is incorporated herein byreference in its entirety.

GOVERNMENTAL INTERESTS

[0002] Subject matter of this application was made in part withgovernment support. The United States Government may retain certainrights in this subject matter.

FIELD OF THE INVENTION

[0003] The present invention is related to the field of diseasediagnosis and treatment. More specifically, the invention is related tothe discovery of single nucleotide polymorphisms (SNPs) and/or mutationsin the Alpha-2-Macroglobulin gene (A2 μM). Included among the A2Mpolymorphisms and/or mutations are those that can be indicative of analtered risk for Alzheimer's Disease (AD).

BACKGROUND OF THE INVENTION

[0004] Alpha-2-Macroglobulin (A2M) is an abundant plasma protein similarin structure and function to a group of proteins calledα-macroglobulins. A2M is also produced in the brain where it bindsmultiple extracellular ligands and is internalized by neurons andastrocytes. In the brain of Alzheimer's disease (AD) patients, A2M hasbeen localized to diffuse amyloid plaques. A2M also binds solubleβ-amyloid and mediates its degradation. An excess of A2M, however, canhave neurotoxic effects. Kovacs, Experimental Gerontology, 35:473-479(2000). Based on genetic evidence, A2M is now recognized as one of thetwo confirmed late onset AD genes. As for the three early onset genes(the amyloid β-protein precursor and the two presenilins) and for theother late onset gene (ApoE), DNA polymorphisms in the A2M geneassociated with AD result in significantly increased accumulation ofamyloid plaques in AD brains. These data support an important role forA2M in AD etiopathology.

[0005] Human A2M is a 720 kDa soluble glycoprotein composed of fouridentical 180 kDa (1451 amino acid) subunits, each of which is encodedby a single-copy gene on chromosome 12. Disulfide bonds and noncovalentinteractions connect the subunits within the tetramer. A2M is oftenreferred to as a panprotease inhibitor, because it entraps and isolatesvirtually any protease from the extracellular environment followed byits degradation. Activation of A2M involves a complex conformationalchange of the tetramer, triggered either by protease cleavage of A2M orby methylamine treatment. Activation of A2M results in the entrapment ofproteases and the exposure of the four receptor binding domains to theextracellular environment.

[0006] In the human A2M tetramer, each subunit contains at least fivebinding sites: the bait region, the internal thiol ester, the receptorbinding site, the Aβ binding site, and the zinc binding site. The baitregion, the internal thiol ester and the receptor binding site have apivotal role in the activation and internalization of A2M. The baitregion in each monomer is located between amino acids 666 to 706, at thecenter of each molecule, and it binds any known protease. The four baitregions in the tetramer are in close contact and are cleaved by thebound proteases, which triggers activation of A2M. This conformationalchange results in a sudden exposure of the four thiol esters betweenCys949 and Glu952, and of the four receptor binding sites, to theextracellular environment.

[0007] The A2M region of chromosome 12 has first been associated with ADin genetic linkage analyses. (See e.g., Scott et al., JAMA, 281:513-514(1999)). Two specific AD-associated polymorphisms have been reported inthe A2M gene: an intronic deletion at exon 18 (18i; see e.g., Matthijsand Marynen, Nucleic Acids Res., 19:5102 (1991)) and a single amino acidsubstitution at position 1000 (1000 V/I; see e.g., Liao et al., Hum.Mol. Genet., 7:1953-1956 (1998)). Both of these polymorphisms were foundto be associated with increased β-amyloid deposition (Myllykangas etal., Ann. Neurol., 46:382-390 (1999)).

[0008] Alzheimer's disease is a devastating neurodegenerative disorderthat affects more than 4 million people per year in the US (Döbeli, H.,Nat. Biotech. 15: 223-24 (1997)). It is the major form of dementiaoccurring in mid to late life: approximately 10% of individuals over 65years of age, and approximately 40% of individuals over 80 years of age,are symptomatic of AD (Price, D. L., and Sisodia, S. S., Ann. Rev.Neurosci. 21:479-505 (1998)). The need for diagnostics and therapeuticsfor AD is manifest.

SUMMARY OF THE INVENTION

[0009] Some aspects of the present invention are described in thenumbered paragraphs below.

[0010] 1. A method for identifying a polymorphism or combination ofpolymorphisms associated with an A2M-mediated disease or disorder,comprising testing one or more polymorphisms in an A2M gene individuallyand/or in combinations for genetic association with an A2M-mediateddisease or disorder, wherein the one or more polymorphisms is/areselected from the group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1,14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e.

[0011] 2. A method for identifying a polymorphism or combination ofpolymorphisms associated with a neurodegenerative disease or disorder,comprising testing one or more polymorphisms in an A2M gene individuallyand/or in combinations for genetic association with a neurodegenerativedisease or disorder, wherein the one or more polymorphisms is/areselected from the group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1,14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e.

[0012] 3. The method of Paragraph 1, wherein the nucleotide at 6i is A,the nucleotide at 12i.1 is G, the nucleotide at 12i.2 is T, thenucleotide at 12e is T, the nucleotide at 14e is C, the nucleotide at14i.2 is C, the nucleotide at 17i.1 is G, the nucleotide at 20e is T,the nucleotide at 20i is G, the nucleotide at 21i is C, the nucleotideat 28i is T and the nucleotide at 30e is C, or the complementartnucleotide thereof.

[0013] 4. The method of Paragraph 2, wherein the nucleotide at 6i is A,the nucleotide at 12i.1 is G, the nucleotide at 12i.2 is T, thenucleotide at 12e is T, the nucleotide at 14e is C, the nucleotide at14i.2 is C, the nucleotide at 17i.1 is G, the nucleotide at 20e is T,the nucleotide at 20i is G, the nucleotide at 21i is C, the nucleotideat 28i is T and the nucleotide at 30e is C, or the complementartnucleotide thereof.

[0014] 5. The method of Paragraph 2, wherein the disease is Alzheimer'sdisease.

[0015] 6. A method of genotyping a cell comprising:

[0016] obtaining from an individual a biological sample containing analpha-2-macroglobulin nucleic acid or portion thereof; and

[0017] determining the identity of one or more nucleotides in saidalpha-2-macroglobulin nucleic acid or portion thereof wherein said oneor more nucleotides are located at a position selected from the groupconsisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i,21i, 28i and 30e.

[0018] 7. The method of Paragraph 6, wherein said alpha-2-macroglobulinnucleic acid is genomic DNA.

[0019] 8. The method of Paragraph 6, wherein said alpha-2-macroglobulinnucleic acid is RNA.

[0020] 9. The method of Paragraph 6, comprising determining the identityof one or more nucleotides at a position selected from the groupconsisting of 6i, 12e, 14i.1 and 20e.

[0021] 10. The method of Paragraph 9, further comprising determining theidentity of one or more nucleotides at position 18i.

[0022] 11. The method of Paragraph 6, comprising determining theidentity of one or more nucleotides at a position selected from thegroup consisting of 6i, 12e, 14i.1 and 21i.

[0023] 12. The method of Paragraph 11, further comprising determiningthe identity of one or more nucleotides at position 18i.

[0024] 13. The method of Paragraph 6, comprising determining theidentity of one or more nucleotides at a position selected from thegroup consisting of 12e, 14i.1 and 21i.

[0025] 14. The method of Paragraph 13, further comprising determiningthe identity of one or more nucleotides at a position selected from thegroup consisting of 18i and 24e.

[0026] 15. The method of Paragraph 6, comprising determining theidentity of one or more nucleotides at a position selected from thegroup consisting of 14i.1, 20e and 21i.

[0027] 16. The method of Paragraph 15, further comprising determiningthe identity of one or more nucleotides at a position selected from thegroup consisting of 18i and 24e.

[0028] 17. The method of Paragraph 6, comprising determining theidentity of one or more nucleotides at a position selected from thegroup consisting of 20e, 21i and 28e.

[0029] 18. The method of Paragraph 17, further comprising determiningthe identity of one or more nucleotides at a position selected from thegroup consisting of 18i and 24e.

[0030] 19. The method of Paragraph 6, comprising determining theidentity of one or more nucleotides at a position selected from thegroup consisting of 6i, 12e, 14i.1 and 21i.

[0031] 20. The method of Paragraph 19, further comprising determiningthe identity of one or more nucleotides at a position selected from thegroup consisting of 18i and 24e.

[0032] 21. A method of genotyping a cell comprising:

[0033] obtaining from an individual a biological sample containing analpha-2-macroglobulin polypeptide or portion thereof; and

[0034] determining the identity of one or more amino acids in saidalpha-2-macroglobulin polypeptide or portion thereof wherein said one ormore amino acids are located at a position selected from the groupconsisting of 14e, 20e and 30e.

[0035] 22. A method of identifying a subject at risk for Alzheimer'sDisease, said method comprising:

[0036] obtaining from said subject a biological sample containing analpha-2-macroglobulin nucleic acid or portion thereof; and

[0037] determining the presence or absence of one or more polymorphismsor mutations in said alpha-2-macroglobulin nucleic acid or portionthereof wherein said one or more polymorphisms or mutations occur at aposition selected from the group consisting of 6i, 12i.1, 12i.2, 12e,14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e.

[0038] 23. The method of Paragraph 22, wherein saidalpha-2-macroglobulin nucleic acid is genomic DNA.

[0039] 24. The method of Paragraph 22, wherein saidalpha-2-macroglobulin nucleic acid is RNA.

[0040] 25. The method of Paragraph 22, wherein the nucleotide at 6i isA, the nucleotide at 12i.1 is G, the nucleotide at 12i.2 is T, thenucleotide at 12e is T, the nucleotide at 14e is C, the nucleotide at14i.2 is C, the nucleotide at 17i.1 is G, the nucleotide at 20e is T,the nucleotide at 20i is G, the nucleotide at 21i is C, the nucleotideat 28i is T and the nucleotide at 30e is C or the complemtarynucleotides thereof.

[0041] 26. The method of Paragraph 22, comprising determining thepresence or absence of one or more polymorphisms at a position selectedfrom the group consisting of 6i, 12e, 14i.1 and 20e.

[0042] 27. The method of Paragraph 26, further comprising determiningthe presence or absence of one or more polymorphisms at position 18i.

[0043] 28. The method of Paragraph 22, comprising determining thepresence or absence of one or more polymorphisms at a position selectedfrom the group consisting of 6i, 12e, 14i.1 and 21i.

[0044] 29. The method of Paragraph 28, further comprising determiningthe presence or absence of one or more polymorphisms at position 18i.

[0045] 30. The method of Paragraph 22, comprising determining thepresence or absence of one or more polymorphisms at a position selectedfrom the group consisting of 12e, 14i.1 and 21i.

[0046] 31. The method of Paragraph 30, further comprising determiningthe presence or absence of one or more polymorphisms at a positionselected from the group consisting of 18i and 24e.

[0047] 32. The method of Paragraph 22, comprising determining thepresence or absence of one or more polymorphisms at a position selectedfrom the group consisting of 14i.1, 20e and 21i.

[0048] 33. The method of Paragraph 32, further comprising determiningthe presence or absence of one or more polymorphisms at a positionselected from the group consisting of 18i and 24e.

[0049] 34. The method of Paragraph 22, comprising determining thepresence or absence of one or more polymorphisms at a position selectedfrom the group consisting of 20e, 21i and 28e.

[0050] 35. The method of Paragraph 34, further comprising determiningthe presence or absence of one or more polymorphisms at a positionselected from the group consisting of 18i and 24e.

[0051] 36. The method of Paragraph 22, comprising determining thepresence or absence of one or more polymorphisms at a position selectedfrom the group consisting of 6i, 12e, 14i.1 and 21i.

[0052] 37. The method of Paragraph 36, further comprising determiningthe presence or absence of one or more polymorphisms at a positionselected from the group consisting of 18i and 24e

[0053] 38. The method of Paragraph 22, comprising determining thepresence or absence of one or more polymorphisms at a position selectedfrom the group consisting of 12e, 12i and 28i.

[0054] 39. The method of Paragraph 38, wherein the nucleotide atposition 12e is T, or the complement thereof, the nucleotide at position21i is A, or the complement thereof and the nucleotide at position 28iis A, or the complement thereof.

[0055] 40. A method of identifying a subject at risk for Alzheimer'sDisease, said method comprising:

[0056] obtaining from said subject a biological sample containing analpha-2-macroglobulin polypeptide or portion thereof; and

[0057] determining the presence or absence of one or more polymorphismsor mutations in said alpha-2-macroglobulin polypeptide or portionthereof wherein said one or more polymorphisms or mutations occur at aposition selected from the group consisting of 14e, 20e and 30e.

[0058] 41. A method of identifying a compound that modulates analpha-2-macroglobulin activity comprising:

[0059] providing a plurality of cells that express the LRP receptor;

[0060] contacting said cells with a candidate compound;

[0061] contacting said cells with an alpha-2-macroglobulin polypeptidecomprising at least one polymorphism or mutation having a positionselected from the group consisting of 14e, 20e, and 30e; and

[0062] identifying a compound that modulates an alpha-2-macroglobulinactivity.

[0063] 42. The method of Paragraph 41, wherein saidalpha-2-macroglobulin activity is an interaction of saidalpha-2-macroglobulin polypeptide with the LRP receptor.

[0064] 43. The method of Paragraph 41, wherein saidalpha-2-macroglobulin activity is the degradation of saidalpha-2-macroglobulin polypeptide.

[0065] 44. The method of Paragraph 41, wherein saidalpha-2-macroglobulin activity is a protease inhibitor activity.

[0066] 45. The method of Paragraph 41, wherein saidalpha-2-macroglobulin activity is the clearance of saidalpha-2-macroglobulin polypeptide.

[0067] 46. The method of Paragraph 41, wherein said cells are contactedwith an alpha-2-macroglobulin polypeptide in the presence of amyloid β.

[0068] 47. The method of Paragraph 46, wherein saidalpha-2-macroglobulin activity is an interaction of amyloid β or saidalpha-2-macroglobulin polypeptide with the LRP receptor.

[0069] 48. The method of Paragraph 47, wherein saidalpha-2-macroglobulin mediates clearance of amyloid β.

[0070] 49. A method of identifying a compound that modulates analpha-2-macroglobulin activity comprising:

[0071] providing an alpha-2-macroglobulin polypeptide comprising atleast one of the polymorphisms or mutations having a position selectedfrom the group consisting of 14e, 20e, and 30e;

[0072] contacting said alpha-2-macroglobulin polypeptide with saidcompound;

[0073] contacting said alpha-2-macroglobulin polypeptide withmethylamine; and

[0074] identifying a compound that modulates an alpha-2-macroglobulinactivity by detecting a modulation in the activation of saidalpha-2-macroglobulin polypeptide. 50. A method of identifying acompound that modulates an alpha-2-macroglobulin activity comprising:

[0075] providing an alpha-2-macroglobulin polypeptide comprising atleast one of the polymorphisms or mutations having a position selectedfrom the group consisting of 14e, 20e, and 30e;

[0076] contacting said alpha-2-macroglobulin polypeptide with saidcompound;

[0077] contacting said alpha-2-macroglobulin polypeptide with amyloid β;and

[0078] identifying a compound that modulates an alpha-2-macroglobulinactivity by detecting a modulation in the formation of a complex ofamyloid β and said alpha-2-macroglobulin polypeptide.

[0079] 51. A method of making a pharmaceutical comprising:

[0080] identifying a compound by a method of any one of Paragraphs 41,49 and 50 incorporating said compound into a pharmaceutical.

[0081] 52. A purified or isolated nucleic acid comprising analpha-2-macroglobulin sequence having a polymorphism or mutation at aposition selected from the group consisting of 6i, 12i.1, 12i.2, 12e,14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i , 28i and 30e, wherein thenucleotide or nucleotide sequence at said position is other than anA2M-1.

[0082] 53. The purified or isolated nucleic acid of Paragraph 52,wherein said alpha-2-macroglobulin sequence is SEQ ID NO: 1 or asequence complementary thereto.

[0083] 54. The purified or isolated nucleic acid of Paragraph 53,wherein the nucleotide or nucleotide sequence at said position is A2M-2.

[0084] 55. The purified or isolated nucleic acid of Paragraph 52,wherein said alpha-2-macroglobulin sequence is selected from the groupconsisting of SEQ ID NOs: 2-8 and said polymorphism of mutation is at aposition selected from the group consisting of 14e, 20e and 30e.

[0085] 56. The purified or isolated nucleic acid of Paragraph 55,wherein the nucleotide or nucleotide sequence at said position is A2M-2.

[0086] 57. The purified or isolated nucleic acid comprising a fragmentof at least 16 consecutive nucleotides of SEQ ID NO: 1 having apolymorphism or mutation at a position selected from the groupconsisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i,21i, 28i and 30e, wherein the nucleotide or nucleotide at said positionis other than an A2M-1 or a sequence complementary thereto.

[0087] 58. The purified or isolated nucleic acid of Paragraph 56,wherein the nucleotide or nucleotide sequence at said position is A2M-2.

[0088] 59. A purified or isolated polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 9-15 having apolymorphism or mutation at a position selected from the groupconsisting of 14e, 20e and 30e, wherein the amino acid at said positionis other than A2M-1.

[0089] 60. The purified or isolated polypeptide of Paragraph 59, whereinthe amino acid at said position is A2M-2.

[0090] 61. A purified or isolated polypeptide comprising a fragment ofan amino acid sequence selected from the group consisting of SEQ ID NOs:9-15 having a polymorphism or mutation at a position selected from thegroup consisting of 14e, 20e and 30e, wherein the amino acid mutation atsaid position is other than A2M-1.

[0091] 62. The purified or isolated polypeptide of Paragraph 61, whereinthe amino acid at said position is A2M-2.

[0092] 63. A recombinant vector comprising the nucleic acid of any oneof Paragraphs 52-58.

[0093] 64. A cultured cell comprising the nucleic acid of any one ofParagraphs 52-58 or the polypeptide of any one of Paragraphs 59-62.

[0094] 65. A cultured cell comprising the recombinant vector ofParagraph 63.

[0095] 66. An isolated or purified antibody that specifically binds tothe polypeptide of any one of Paragraphs 59-62.

[0096] 67. The antibody of Paragraph 66, wherein said antibody ismonoclonal. 68. A method of expressing an alpha-2-macroglobulinpolypeptide comprising:

[0097] providing a construct comprising a promoter operably linked to analpha-2-macroglobulin nucleic acid having a polymorphism or mutation ata position selected from the group consisting of 14e, 20e and 30e,wherein the nucleotide at said position is other than an A2M-1; and

[0098] expressing said alpha-2-macroglobulin from said construct.

[0099] 69. The method of Paragraph 68, wherein said nucleotide at saidposition is A2M-2.

BRIEF DESCRIPTION OF THE DRAWINGS

[0100] The Figure shows a nucleotide sequence of a portion of chromosome12 that includes the genomic sequence of A2M that has been annotated toinclude the locations of exons as well as the names and locations of thepolymorphisms and/or mutations described herein. The name of thepolymorphism and/or mutation as well as the corresponding nucleotidechange(s) are indicated at positions above the A2M gene sequence. Thenucleotide sequence provided in the Figure is from the University ofCalifornia at Santa Cruz draft human genome sequence build 12 forchromosome positions 9007566-8918942 as is available atwww.genome.ucsc.edu. The sequence presented is that of the “minus”strand in the sense that it is the complement of the strand that extends5′→3′ from the p terminus to the centromere of chromosome 12. Thesequence is, however, presented as the “sense” strand for the A2M gene.The sense strand refers to that strand of a double stranded nucleic acidmolecule associated with a gene that has the sequence of the mRNA thatencodes the amino acid sequence. This sequence also corresponds tonucleotides 1-88624 of NCBI Accession Number AC007436 (SEQ ID NO: 1).

DETAILED DESCRIPTION OF THE INVENTION

[0101] Several single nucleotide polymorphisms (SNPs) and/or mutationsof A2M gene have been discovered. Specifically, several novel SNPsand/or mutations were found in patients suffering from Alzheimer'sDisease (AD). These SNPs and/or mutations are referred to as: 6i, 12i.1,12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e. Thelocation of each of these SNPs and/or mutations on the A2M gene (HumanGenome Project Gene Locus chr12: 9007566-8918942 (minus strand);including a section of human chromosome 12 the sequence of which isprovided in National Center for Biotechnology Information (NCBI)Accession Number NT009702, incoporated herein by reference, and alsopresent as nucleotides 1-88624 of NCBI Accession Number AC007436,incorporated herein by reference) (SEQ ID NO: 1) is identified in Table1 and the Figure. Provided herein are polymorphisms in the region ofchromosome 12 surrounding and including the A2M gene. Thus, thepolymorphisms provided herein include polymorphisms in exons, introns orintervening sequences, intergenic regions and gene upstream anddownstream regions, such as, for example, gene expression regulatoryregions.

[0102] A particular polymorphism, depending on the nature and locationof the polymorphism(s) in a gene allele, can play various roles in themanifestation of a disease condition or disorder. A polymorphism thatgives rise to a particular variant phenotype can produce its effect(s),for example, at the level of RNA or protein. Effects on RNA includealtered splicing, stability, editing and expression. Effects on theprotein include altered protein function, folding, transport,localization, stability and expression. Polymorphisms located in the 5′untranslated region of the gene may alter the activity of an element ofthe gene promoter and change the expression of the mRNA (e.g., level,pattern and/or timing of expression). Polymorphisms located in intronsmay alter RNA stability, editing, splicing, etc. Polymorphisms locatedin the 3′ untranslated region may influence polyadenylation,transcription and/or mRNA stability. Silent alterations in the codingregion of a gene may affect codon usage and/or splicing. Changes in anencoded amino acid sequence, e.g., deletions and insertions, may affectprotein function by increasing or decreasing a native function orbringing about an altered function.

[0103] The first column of Table 1 provides a name for each of the novelSNPs or mutations described herein. The name of the SNP or mutation(i.e., the polymorphism designation) corresponds to its general locationin the A2M gene. For example, 14e refers to a SNP present in exon 14 ofthe A2M gene whereas 12i.1 refers to a SNP present in intron 12 of theA2M gene. The number to the right of the decimal point in 12i.1indicates that this SNP is one of multiple SNPs found in intron 12.Table 1 also provides the location of each SNP with reference to SEQ IDNO: 1 (SEQ ID NO: 1 is the sequence of nucleotides 1-88624 of NCBIAccession Number AC007436, which contains the sequence of an A2M gene)and the nucleotide change(s) caused by each SNP or mutation. Inparticular, for each of the polymorphisms and/or mutations set out inTable 1, except for the 14i.1 mutation, the nucleotide to the left ofthe arrow in column 4 represents the nucleotide present in SEQ ID NO: 1at the position indicated in column 2 of Table 1 (A2M-1). The nucleotideto the right of the arrow represents the nucleotide substitution thatoccurs at this position (A2M-2). For example, the A2M-1 allele of SNP 6icomprises a C at nucleotide position 37221 of NCBI Accession NumberAC007436. The A2M-2 allele of SNP 6i comprises an A at nucleotideposition 37221 of NCBI Accession Number AC007436. For the 14i.1mutation, the A2M-2 allele comprises an insertion of the nucleotides“AAG” immediately following the nucleotide position indicated in column2 of Table 1.

[0104] When reference is made herein to a SNP or mutation (as designatedin column 1) with respect to a cDNA or any other contiguous nucleic acidsequence which encodes A2M, the location of the SNP or mutation withrespect to a specific cDNA or A2M coding sequence is set out in column 3of Table 1. Accordingly, the location of a SNP and or mutation in aparticular cDNA or A2M coding sequence can be determined with referenceto-Table 1, column 3.

[0105] In cases where the SNP or mutation results in an amino acidchange, the amino acid change and position are noted. The amino acid tothe left of the arrow in column 5 represents the A2M-1 amino acid at theposition indicated. The amino acid to the right of the arrow representsthe A2M-2 amino acid at the position indicated. The Figure provides anannotated A2M gene sequence which shows each of the SNPs and/ormutations listed in Table 1, including both the A2M-1 alleles,represented by the nucleotides of SEQ ID NO: 1, and the A2M-2 alleles,represented by the nucleotides listed immediately above SEQ ID NO: 1.Accordingly, the locations of nucleotide or amino acid sequencepolymorphisms set forth in Table 1 are referred to by the polymorphismdesignation (i.e., as set forth in column 1 of Table 1) with referenceto a location corresponding to the nucleotide or amino acid position asset forth in columns 2 and 5 of Table 1, respectively.

[0106] Generally, when a polymorphism designation, for example, 6i, isreferred to herein, it is used to specify a position or location withinan A2M gene, cDNA, mRNA, hnRNA or protein sequence, without regard tothe particular nucleotide or amino acid that may be present at theposition. The nucleotide or amino acid at the specified location of theA2M gene or A2M protein can be any nucleotide or amino acid unless aparticular nucleotide or amino acid is specified. TABLE 1 Novel SNPs andMutations Associated with Alzheimer's Disease Location with reference toNCBI SNP/ Accession Number AC007436 Location with reference to codingNucleotide Amino Acid Change (with Mutation (SEQ ID NO: 1) nucleotidesequences (e.g. cDNAs) Change(s) reference to SEQ ID NO: 9) 6i 174 bpdownstream of exon 6 C→A nucleotide position 37221 12e exon 12Nucleotide positions: 1339 of SEQ ID NOs: 3 C→T Y→Y nucleotide position45269 and 5; and 1338 of SEQ ID NO: 7 Silent effect 12i.1 152 bpupstream of exon 12 C→G nucleotide position 45088 12i.2 115 bp upstreamof exon 12 A→T nucleotide position 45125 14e exon 14 Nucleotidepositions: 1730 of SEQ ID NOs: 3 T→C C→R nucleotide position 47519 and5; and 1729 of SEQ ID NO: 7 Amino acid position 563 14i.1 136 bpdownstream of exon 14 insertion nucleotide position 47669 of AAG 14i.2151 bp downstream of exon 14 A→C nucleotide position 47684 17i.1 240 bpupstream of exon 18 C→G nucleotide position 53095 20e exon 20 Nucleotidepositions: 2574 of SEQ ID NOs: 3 C→T A→V nucleotide position 56493 and5; 2573 of SEQ ID NO: 7; and 38 of SEQ Amino acid position 844 ID NO: 420i 27 bp downstream of exon 20 C→G nucleotide position 56586 21i 2 bpupstream of exon 21 T→C nucleotide position 56887 28i 55 upstream ofexon 29 G→T nucleotide position 72076 30e exon 30 Nucleotide positions:3912 of SEQ ID NOs: 3 T→C F→L nucleotide position 74154 and 5; 3911 ofSEQ ID NO: 7; and 1376 of SEQ Amino acid position 1290 ID NO: 4

[0107] Table 2 provides a list of additional SNPs and mutations andtheir position on the A2M gene. The Figure also shows the positions ofeach of the SNPs and mutations listed in Table 2 as well as thenucleotide change (A2M-2) that is associated with the SNP and/ormutation. TABLE 2 Additional SNPs and Mutations Associated withAlzheimer's Disease A2M Gene Sequence Database Chromosome 12 CoordinateNCBI SNP Identifier Coordinate Accession AC007436 rs226379 8976642 30925rs226380 8976530 31037 rs226381 8975616 31951 rs3080605 8975391 32176rs226382 8974334 33233 rs2302666 8973921 33646 rs2477 8973853 33714rs226383 8973003 34564 rs226384 8971704 35863 rs226385 8971288 36279rs226386 8970784 36783 rs226387 8969302 38265 rs226388 8968337 39230rs226389 8967964 39603 rs1049134 8964919 42648 rs226390 8964765 42802rs226391 8964411 43156 rs226392 8964312 43255 rs226393 8963888 43679rs226394 8963091 44476 rs226395 8962840 44727 rs226396 8962283 45284rs226397 8961951 45616 rs226398 8961373 46194 rs226399 8959102 48465rs226400 8958524 49043 rs226401 8958516 49051 rs226402 8957932 49635rs226403 8957810 49757 rs226404 8956453 51114 rs226405 8956290 51277rs1800434 8955640 51927 rs226406 8954411 53156 rs226407 8953836 53731rs226408 8953258 54309 rs226409 8953062 54505 rs226410 8952700 54867rs113973 8952324 55243 rs2277412 8952004 55563 rs1049143 8951935 55632rs2277413 8951903 55664 rs3180392 8951879 55688 rs3210107 8951879 55688rs226411 8951178 56389 rs226412 8949081 58486 rs226413 8948804 58763rs2889706 8948741 58826 rs2111023 8948292 59275 rs226414 8947972 59595rs2193006 8944647 62920 rs1800433 8940408 67159 rs3168556 8940325 67242rs1805651 8939695 67872 rs1805652 8938629 68938 rs1805653 8938188 69379rs2377682 8938095 69472 rs1805654 8937686 69881 rs1805678 8937227 70340rs1805655 8936701 70866 rs1805656 8936688 70879 rs1805679 8936686 70881rs3026223 8936527 71040 rs1805657 8936491 71076 rs1805680 8936426 71141rs1805658 8936355 71212 rs1805659 8936312 71255 rs3026224 8936205 71362rs2300147 8936088 71479 rs2300148 8936081 71486 rs1805681 8935925 71642rs1805682 8935844 71723 rs1805683 8935145 72422 rs1805660 8935115 72452rs1805661 8935018 72549 rs3080599 8934757 72810 rs1805684 8934307 73260rs3026225 8934282 73285 rs1805662 8934281 73286 rs1805685 8933979 73588rs1805663 8932010 75557 rs1805664 8930343 77224 rs1805665 8930160 77407rs1805666 8930154 77413 rs3026226 8930105 77462 rs3026227 8929855 77712rs1805686 8929764 77803 rs3026228 8929693 77874 rs3180682 8928606 78961rs1805687 8928558 79009 rs1049985 8928436 79131 rs3190224 8928425 79142rs1805688 8928157 79410 rs1805667 8928023 79544 rs3026229 8927957 79610

[0108] It will be appreciated that the nomenclature for thepolymorphisms and/or mutations used in the Figure and in Tables 1 and 2refers to the location of the polymorphism and/or mutation disclosedherein. Accordingly, the use of a polymorphism or mutation name (ordesignation), such as 6i, 14e, or rs226381 indicates a polymorphicposition in the reference nucleotide or amino acid sequence and notnecessarily the identity of the nucleotide or amino acid change. Thenucleotide and amino acid changes indicated in the Figure and in Table 1correspond to one of many changes which can occur at the location of thepolymorphism and/or mutation.

[0109] The reference nucleic acid sequence is provided by SEQ ID NO:1which corresponds to nucleotides 1-88624 of NCBI Accession NumberAC007436. It will be appreciated that a nucleic acid corresponding to anA2M coding sequence (SEQ ID NO: 2) can be constructed by joining theexons at the splice sites listed for nucleotide sequence region 1-88624as provided in the header section of NCBI Accession Number AC007436.Additionally, a number of cDNA variants of A2M are also available. ThesecDNAs, some of which encode variant polypeptides, are provided as SEQ IDNOs: 3-8. Variant A2M polypeptide sequences are provided as SEQ ID NOs:9-15.

[0110] In view of the above, it will be appreciated that, although eachof the novel SNPs and/or mutations disclosed herein are described withreference to SEQ ID NO: 1 (as well as SEQ ID NOs:2-15), each of theseSNPs and/or mutations can occur in the context of nucleic acid sequencevariants. For example, in addition to one or more of the SNPs disclosedherein, SNPs and/or mutations previously described for A2M (e.g. SNPsand/or mutations described in Table 2) may occur within SEQ ID NO: 1 (aswell as SEQ ID NOs:2-15). Such nucleic acids having both one or more ofthe SNPs and/or mutations described herein and one or more known orpreviously described SNPs and/or mutations for A2M are contemplated bythe present invention. Furthermore, A2M genes that have one or more ofthe SNPs and/or mutations described herein and which are altered fromSEQ ID NO: 1 (as well as SEQ ID NOs:2-15) or known variants thereof asresult from one or more sequencing errors are also contemplated by thepresent invention. As used herein, the term “mutation” means nucleotidevariations that are not limited to single nucleotide substitution. Forexample, a mutation includes, but is not limited to, the insertion ofone or more bases, the deletion of one or more bases, or an inversion ofmultiple bases.

[0111] In view of the above, as used herein, “A2M”, “A2M gene” or “A2Mgenomic nucleic acid”, when used with reference to SEQ ID NO: 1, meansthe nucleic acid sequence of SEQ ID NO: 1 or portions thereof as well asany nucleic acid variants which include one or more SNPs and/ormutations, such as those described in Table 2 and the Figure. Similarly,“A2M cDNA”, “A2M coding sequence” or “A2M coding nucleic acid”, whenused with reference to SEQ ID NOs: 2-8, means the nucleic acid sequencesof SEQ ID NOs: 2-8 or portions thereof as well as nucleic acid variantswhich include one or more SNPs and/or mutations, such as those describedin Table 2 and the Figure. With respect to polypeptides “A2M”, “A2Mpolypeptide” or “A2M protein”, when used with reference to SEQ ID NOs:9-15, means the amino acid sequence of SEQ ID NOs: 9-15 or portionsthereof as well as amino acid sequence variants which are encoded bynucleic acids which include one or more SNPs and/or mutations, such asthose described in Table 2, and the Figure and which effect thepolypeptide encoded by the A2M coding sequence.

[0112] According to some aspects of the present invention, A2M includesnucleotide sequences having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, 90%, 89%, 88%, 87%, 86%, or 85% sequence identity to SEQ IDNO: 1 as determined by BLASTN with default parameters (Altschul et al,(1990) J. Mol. Biol. 215: 403, incorporated herein by reference in itsentirety). In other aspects of the present invention, A2M codingsequence includes nucleotides sequences having at least 99%, 98%, 97%,96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or 85% sequenceidentity to any one of SEQ ID NOs: 2-8 as determined by BLASTN version2.0 with default parameters (Altschul et al, (1990) J. Mol. Biol. 215:403, incorporated herein by reference in its entirety). In still otheraspects of the present invention, A2M includes polypeptide sequenceshaving at least 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%,84%, 83%, 82%, 81%, or 80% sequence identity or similarity to any one ofSEQ ID NOs: 9-15 as determined by FASTA version 3.0t78 with defaultparameters (Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA, 85:2444, incorporated herein by reference in its entirety).

[0113] As used in connection with any one of the polymorphisms and/ormutations disclosed herein, A2M-1 refers to the nucleotide or nucleotidesequence of SEQ ID NO: 1 which is present at the location of thepolymorphism or mutation. As used in connection with any one of thepolymorphisms and/or mutations disclosed herein, A2M-2 refers to thenucleotide change, nucleotide insertion or nucleotide deletion indicatedin the Figure and/or in Table 1 which is present at the location of thepolymorphism or mutation. As used in connection with any one of thepolymorphisms and/or mutations disclosed herein, A2M-1 refers to theamino acid of SEQ ID NO: 9 which is present at the location of thepolymorphism or mutation. As used in connection with any one of thepolymorphisms and/or mutations disclosed herein, A2M-2 refers to theamino acid change indicated in the Figure and/or in Table 1 which ispresent at the location of the polymorphism or mutation.

[0114] Polymorphisms can serve as genetic markers. A genetic marker is aDNA segment with an identifiable location in a chromosome. Geneticmarkers may be used in a variety of genetic studies such as, forexample, locating the chromosomal position or locus of a DNA sequence ofinterest, identifying genetic associations of a disease, and determiningif a subject is predisposed to or has a particular disease. Because DNAsequences that are relatively close together on a chromosome tend to beinherited together, tracking of a genetic marker through generations ina family and comparing its inheritance to the inheritance of another DNAsequence of interest can provide information useful in determining therelative position of the DNA sequence of interest on a chromosome.Genetic markers particularly useful in such genetic studies arepolymorphic. Such markers also may have an adequate level ofheterozygosity to allow a reasonable probability that a randomlyselected person will be heterozygous.

[0115] The polymorphisms provided herein in the region of chromosome 12surrounding and including the A2M gene include single nucleotidepolymorphisms (SNPs). SNPs have use as genetic markers, for example, infine genetic mapping and genetic association analysis, as well aslinkage analysis [see, e.g., Kruglyak (1997) Nature Genetics 17:21-24].Combinations of SNPs (which individually occur about every 100-300bases) can also yield informative haplotypes. Also provided herein, arepolymorphisms of the A2M gene and surrounding region of chromosome 12that are associated, individually and/or in combination, with aneurodegenerative disease, such as, for example, Alzheimer's disease.

[0116] Based on the discovery of association between SNPs describedherein, individually and/or in combinations (haplotypes), with AD,additional markers associated with AD may now be identified usingmethods as described herein and known in the art. The availability ofadditional markers is of particular interest in that it will increasethe density of markers for this chromosomal region and can provide abasis for identification of an AD DNA segment or gene in the region ofchromosome 12. An AD DNA segment or gene may be found in the vicinity ofthe marker or set of markers showing the highest correlation with AD.Furthermore, the availability of markers associated with AD makespossible genetic analysis-based methods of determining a predispositionto or the occurrence of AD in an individual by detection of a particularallele.

[0117] Polymorphisms of the A2M gene region of chromosome 12 providedherein may be analyzed individually and in combinations, e.g.,haplotypes, for genetic association with any disease or disorder. In aparticular example, the disease is a neurodegenerative disease, such as,for example, AD. Thus, also provided herein are methods of identifyingpolymorphisms associated with diseases and disorders. The methodsinvolve a step of testing polymorphisms of the A2M gene, and/orsurrounding region of chromosome 12, and in particular the polymorphismsprovided herein, individually or in combination, e.g., haplotypes, forassociation with a disease or disorder. For example, the polymorphismsprovided herein can be tested individually, in combinations of theprovided polymorphisms, or in combinations with other previouslydescribed polymorphisms (e.g., polymorphisms listed in Table 2). Theanalysis or testing may involve genotyping DNA from individuals affectedwith the disease or disorder, and possibly also from related orunrelated individuals, with respect to the polymorphic marker andanalyzing the genotyping data for association with the disease ordisorder using methods described herein and/or known to those of skillin the art. For example, statistical analysis of the data may involve achi-squared or Fisher's exact test and may be conducted in conjunctionwith a number of programs, such as the transmission disequilibrium test(TDT), affected family based control test (AFBAC) and the haplotyperelative risk test (HRR). Case-control strategies can be applied to thetesting, as can, for example, TDT approaches.

[0118] Several embodiments of the invention have biotechnological,diagnostic, and therapeutic use. For example, the nucleic acids andproteins described herein can be used as probes to isolate morepolymorphic and/or mutant A2M genes, to detect the presence or absenceof wild type or polymorphic and/or mutant A2M proteins in an individual,and these molecules can be incorporated into constructs for preparingrecombinant polymorphic and/or mutant A2M proteins or used in methods ofsearching or identifying agents that modulate A2M levels and/oractivity, for example, candidate therapeutic agents. The sequences ofthe nucleic acids and/or proteins described herein can also beincorporated into computer systems, used with modeling software so as toenable rational drug design. Information obtained from genotypingmethods provided herein can be used, for example, in computer systems,in pharmacogenomic profiling of therapeutic agents to predicteffectiveness of an agent in treating an individual for aneurodegenerative disease such as AD. The nucleic acids and/or proteinsdescribed herein can also be incorporated into pharmaceuticals and usedfor the treatment of neuropathies, such as Alzheimer's Disease (AD).

[0119] Accordingly, some embodiments of the invention include isolatedor purified nucleic acids comprising, consisting essentially of, orconsisting of an A2M gene, cDNA or mRNA with one or more of the SNPsand/or mutations described in Table I or a fragment of said A2M gene,cDNA or mRNA, wherein said fragment contains at least 9, at least 16 orat least 18 consecutive nucleotides of the polymorphic or mutant A2Mgene, cDNA or mRNA but including at least one of the SNPs and/ormutations in Table 1. Isolated or purified nucleic acids that arecomplementary to said A2M nucleic acids and fragments thereof are alsoembodiments.

[0120] Some nucleic acid embodiments for example, include genomic DNA,RNA, and cDNA encoding the polymorphic and/or mutant A2M proteins orfragments thereof. Methods for obtaining such nucleic acid sequences arealso embodiments. The nucleic acid embodiments can be altered, mutated,or changed such that the alteration, mutation, or change results in aconservative amino acid replacement. These altered or changed nucleicacids are equivalent to the nucleic acids described herein. In somecontexts, the term “consisting essentially of” is used to includenucleic acids having the changes or alterations above.

[0121] Vectors having the nucleic acids above, including expressionvectors, and cells containing said nucleic acids and vectors are alsoembodiments. Methods of making these constructs and cells are aspects ofthe invention, as well. Other embodiments of the invention includegenetically altered organisms that express the polymorphic and/or mutantA2M transgenes or polymorphic portions thereof (e.g., mutant A2Mtransgenic or knockout animals). Methods of making such organisms arealso aspects of the invention. Transgenic animals that are contemplated(particularly non-human animals) can be used, for example, inelucidating disease processes and/or identifying therapeutic agents.

[0122] Some polypeptide embodiments of the invention include isolated,enriched, recombinant or purified polypeptides consisting of, consistingessentially of, or comprising the complete amino acid sequences (orportions thereof containing the polymorphic amino acid change) of thepolymorphic and/or mutant A2M proteins described herein. (See Table 1,which includes the nucleotide polymorphisms of the A2M gene codingsequence that result in corresponding amino acid changes in the A2Mpolypeptide sequence. Additionally, Table 1 sets out the identity andlocation of the amino acid substitution with respect to a reference A2Mpolypeptide sequence). Other polypeptide embodiments are equivalents tothe polymorphic and/or mutant A2M proteins described herein in that saidequivalent molecules have conservative amino acid substitutions. In somecontexts, the term “consisting essentially of” is used to includepolypeptides having such conservative amino acid substitutions.Embodiments also include isolated, enriched, recombinant or purifiedfragments of the polymorphic and/or mutant A2M proteins at least 3 aminoacids in length so long as said fragments contain at least one of theamino acid polymorphisms and/or mutants described herein (See Table 1).Additional embodiments concern methods of preparing the polypeptides andpeptides described herein and, in some preparative methods, chemicalsynthesis and/or recombinant techniques are used.

[0123] Embodiments of the invention also include antibodies directed tothe mutant and/or polymorphic A2M proteins. Preferably, said antibodiesspecifically interact with the mutant and/or polymorphic A2M proteinsand can be used to differentiate wild-type A2M proteins (e.g., A2Mproteins having a reference sequence of amino acids and/or that are mostprevalent in the population or in a particular study) from polymorphicand/or mutant A2M proteins. The antibody embodiments can be monoclonalor polyclonal and approaches to manufacture both types of antibodies,which are specific for the polymorphic and/or mutant A2M proteins aredisclosed.

[0124] Approaches to rational drug design are also provided in thisdisclosure, and these methods can be used to identify molecules thatinteract with the polymorphic and/or mutant A2M proteins or fragmentsthereof. Molecules that interact with the polymorphic and/or mutant A2Mproteins or fragments thereof are referred to as “binding partners”.Preferred binding partners modulate (e.g., increase or decrease) theactivity of the polymorphic and/or mutant A2M proteins or fragmentsthereof The various activities of the polymorphic and/or mutant A2Mproteins or fragments thereof can include, but are not limited to, theability to bind proteases, bind amyloid-β, bind a receptor (e.g., theLRP receptor), bind zinc, and the ability to form a tetramer. Severalcomputer-based methodologies are discussed, which involvethree-dimensional modeling of the polymorphic and/or mutant A2M proteinsor fragments thereof and suspected binding partners (e.g., antibodies,proteases, amyloid-P, zinc, and the LRP receptor).

[0125] Several A2M characterization assays are also described. Theseassays test the functionality of a polymorphic and/or mutant A2M proteinor fragment thereof and can identify agents that modulate the activityand/or expression of such proteins, including, for example, bindingpartners that interact with said molecules. Agents that modulate theactivity of a wild-type or polymorphic or mutant A2M, for example, canbe identified using an A2M characterization assay and moleculesidentified using these methods can be incorporated into medicaments andpharmaceuticals, which can be provided to subjects in need of treatmentor prevention of neuropathies, including AD.

[0126] Some functional assays involve the use of multimeric polymorphicand/or mutant A2M proteins or fragments thereof and/or binding partners,which are disposed on a support, such as a resin, bead, lipid vesicle orcell membrane. These multimeric agents are contacted with candidatebinding partners and the association of the binding partner with themultimeric agent is determined. Successful binding agents can be furtheranalyzed for their effect on A2M function in other types of cell basedassays. One such assay evaluates internalization of a protease oramyloid β. Other types of characterization assays involve molecularbiology techniques designed to identify protein-protein interactions(e.g., two-hybrid systems).

[0127] The diagnostic embodiments of the invention (including diagnostickits) are designed to identify individuals at risk of acquiring AD orindividuals that have a predilection for AD. Nucleic acid and proteinbased diagnostics are provided. Some of these diagnostics identifyindividuals at risk for acquiring AD by detecting a particularnucleotide or amino acid polymorphism and/or mutation or combinations ofpolymorphisms and/or mutations, for example a haplotype, in an A2M geneor A2M protein. Other diagnostic approaches are concerned with thedetection of aberrant amounts or levels of expression of polymorphic ormutant A2M RNA or A2M protein. The polymorphisms and/or mutations,levels of expression of polymorphic or mutant A2M RNAs or proteins canbe recorded in a database, which can be accessed to identify a type ofAD, a suitable treatment., and subjects for which further genotypingshould be investigated. It is contemplated that many other SNPs and/ormutations, which are predictive of AD, can be found in subjectsidentified as already having at least one SNP and/or mutation describedherein.

[0128] Accordingly, a method of identifying an individual having analtered risk for AD is provided, wherein a biological sample containingnucleic acid is obtained from an individual, and the sample is analyzedto determine the nucleotide identity of at least one novel SNP and/ormutation, such as at least one SNP and/or mutation selected from thegroup consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1,20e, 20i, 21i, 28i and 30e. The presence or absence of a particularnucleotide or nucleotide sequence at the location of any one of theseSNPs and/or mutations can indicate an altered risk of AD. Additionally,the nucleotide identity information obtained from the analysis ofcombinations of SNPs and/or mutations can further indicate an alteredrisk of AD. The biological sample can also be analyzed to determine thenucleotide identity of publicly available SNPs and/or mutations.Nucleotide identity information obtained from the analysis of publiclyavailable SNPs and/or mutations in combination with novel SNPs disclosedherein, such as at least one SNP and/or mutation selected from the groupconsisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i,21i, 28i and 30e, can indicate an altered risk for AD. The analysis caninclude an association study (e.g., a family study) and/or haplotypeanalysis.

[0129] Also provided are methods of identifying polymorphisms associatedwith a disease or disorder. The novel SNPs and/or mutations describedherein, such as a SNP and/or mutation selected from the group consistingof 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28iand 30e, can be analyzed separately or in combinations to identifyassociation with any A2M-mediated disease or disorder. The polymorphismscan be analyzed to identify association with neurodegenerative diseases.For example, a single or combinations of novel SNPs and/or mutations canbe checked for association with neurodegenerative disorders or otherdiseases having a relationship to the A2M gene using methods well knownin the art, such as those described herein.

[0130] For example, the genotype of individuals with respect to one ormore polymorphisms and/or mutations selected from the group consistingof 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28iand 30e can be compared between individuals that have AD or a particulardisease or a family history of the disease and individuals that do nothave the disease or a family history of the disease so as to identify apolymorphism or combination of polymorphisms that associate with adisease or disorder, such as a neurodegenerative disease or disorder,for example AD. Additionally, since there are many different genotypesthat can be associated with AD, individuals with AD having one genotypecan be compared with individuals with AD having another genotype toidentify the presence of a novel SNP and/or mutation. In one embodimentof the invention, the information and analysis above can be recorded ona database and the comparisons can be performed by a computer systemaccessing said database. Thus, by virtue of the fact that at least oneSNP and/or mutation selected from the group consisting of 6i, 12i.1,12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e hasbeen identified in an individual or a family, the nucleic acids andproteins isolated or purified from said individuals becomes a novel toolwith which more SNPs and mutations associated with AD can be identified.

[0131] In yet another aspect of the present invention, the informationgained from analyzing biological samples obtained from one or moreindividuals to determine the nucleotide identity of at least one novelSNP and/or mutation described herein, such as the SNPs and/or mutationsselected from the group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1,14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e, can be used in fine chromosomemapping of chromosome 12, in genetic association studies, inpharmacogenetic profiling and pharmacogenetic-based treatment programsand in the search for a gene responsible for AD or other AD-associatedgenes.

[0132] Also provided herein are methods of genotyping an individualcomprising obtaining a nucleic acid sample from an individual anddetermining the nucleotide identity of at least one novel SNP and/ormutation described herein, such as at least one SNPs and/or mutationsselected from the group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1,14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e. In a particular embodiment,the nucleotide identity of more than one novel polymorphism and/ormutation is determined. Accordingly, a set of novel polymorphisms and/ormutations can be analyzed to determine the nucleotide identity for eachpolymorphism and/or mutation in the entire set. The set of polymorphismsand/or mutations can also include polymorphisms and/or mutations thatare publicly available as well as novel polymorphisms and/or mutations.Determination of the nucleotide identities for sets of polymorphismsand/or mutations as described above provides a method for determiningthe haplotype of an individual.

[0133] Also provided herein are methods of confirming a phenotypicdiagnosis of a disease or disorder which include a step of detecting innucleic acid obtained from a subject diagnosed with a disease ordisorder the presence or absence of one or more polymorphisms and/orselected from the group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1,14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e, wherein the presence of theone or more polymorphisms, individually and/or in combination, confirmsa phenotypic diagnosis of the disease or disorder. In a particularembodiment of these methods, the disease or disorder is an A2M-mediateddisease disorder. In one embodiment, the disease or disorder is aneurodegenerative disease or disorder, such as, for example, AD. Forexample, the disease may be Alzheimer's disease with an onset age ofgreater than or equal to about 50 years, or greater than or equal toabout 60 years, or greater than or equal to about 65 years. In anotherembodiment of the methods of confirming a phenotypic diagnosis of aneurodegenerative disease or disorder, the method further includes astep of detecting in nucleic acid obtained from the subject the presenceor absence of one or more polymorphisms of at least one different geneallele associated with neurodegenerative disease. In a particularembodiment, the at least one different gene allele is an APOE4 allele.

[0134] Further provided are methods of treating a subject manifesting anAlzheimer's disease phenotype. Certain ambiguous phenotypes, e.g.,dementia, manifested in AD also occur in connection with other diseasesand conditions which may be treated using drugs and other treatmentsthat are different from drugs and methods used to treat AD. Genotypingof polymorphisms of the A2M gene region described herein, and optionallyother AD-associated markers, in subjects manifesting such an ADphenotype(s) permits confirmation of AD phenotypic diagnoses and assistsin distinguishing between AD and other possible diseases or disorders.Once an individual is genotyped as having or being predisposed to AD, heor she may be treated with any known methods effective in treating AD.

[0135] Accordingly, methods of treating a subject manifesting anAlzheimer's disease phenotype provided herein include steps of

[0136] (a) determining the nucleotide identity, in a nucleic acidobtained from the subject, of one or more polymorphisms selected fromthe group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1,20e, 20i, 21i, 28i and 30e, wherein the presence of a particularnucleotide or nucleotides at the one or more polymorphisms, individuallyand/or in combination, is indicative of the occurrence of Alzheimer'sdisease in a subject; and

[0137] (b) selecting and/or administering a treatment that is effectivefor treatment of Alzheimer's disease.

[0138] The pharmaceutical embodiments of the invention includemedicaments containing an agent, for example, a binding partners thatmodulates the activity of wild-type or polymorphic or mutant A2M. Thesemedicaments can be prepared in accordance with conventional methods ofgalenic pharmacy for administration to organisms in need of treatment. Atherapeutically effective amount of agent, for example, a bindingpartner (e.g., an amount sufficient to modulate the function of awild-type or polymorphic or mutant A2M) can be incorporated into apharmaceutical composition with or without a carrier. Routes ofadministration of the pharmaceuticals of the invention include, but arenot limited to, topical, transdermal, parenteral, gastrointestinal,transbronchial, and transalveolar. These pharmaceuticals can be providedto subjects in need of treatment for neurodegenerative diseases, inparticular AD. The section below describes several of the nucleic acidembodiments of the invention.

[0139] A2M Nucleic Acids

[0140] The A2M nucleotide sequences of the invention include: (a) thenucleotide sequence provided in NCBI Accession Number AC007436nucleotide positions 1-88624, incorporated herein by reference in itsentirety (SEQ ID NO: 1), or a portion thereof, as modified by anucleotide(s) change at least one SNP and/or mutation selected from thegroup consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1,20e, 20i, 21i, 28i and 30e as indicated in the Figure and/or in Table 1;(b) nucleotide sequences encoding amino acid sequences (a sequenceformed by the joining the exons of the genomic sequence provided in NCBIAccession Number AC007436 between nucleotide positions 31033 and 79197(SEQ ID NO: 2), or A2M, mRNA or cDNA sequences (e.g., SEQ ID NOs: 3-8)as modified by a nucleotide(s) change at least one SNP and/or mutationselected from the group consisting of 12e, 14e, 20e, and 30e asindicated in the Figure and/or in Table 1; (c) the nucleotide sequenceprovided in SEQ ID NO: 1, or a portion(s) thereof, wherein thenucleotide at a position corresponding to 37221 is A, T or G, thenucleotide at a position corresponding to 45269 is T, A or G, thenucleotide at a position corresponding to 45088 is G, A or T, thenucleotide at a position corresponding to 45125 is T, C or G, thenucleotide at a position corresponding to 47519 is C, A or G, thenucleotide at a position corresponding to 47684 is C, G or T, thenucleotide at a position corresponding to 53095 is G, A or T, thenucleotide at a position corresponding to 56493 is T, A or G, thenucleotide at a position corresponding to 56586 is G, A or T, thenucleotide at a position corresponding to 56887 is C, G or A, thenucleotide at a position corresponding to 72076 is T, A or C, thenucleotide at a position corresponding to 74154 is C, A or G, and/or thesequence of AAG occurs between nucleotides at positions corresponding topositions 47669 and 47670; and (d) the nucleotide sequence provided inSEQ ID NO: 1, or a portion(s) thereof, wherein the nucleotide at aposition corresponding to 37221 is A, the nucleotide at a positioncorresponding to 45269 is T, the nucleotide at a position correspondingto 45088 is G, the nucleotide at a position corresponding to 45125 is T,the nucleotide at a position corresponding to 47519 is C, the nucleotideat a position corresponding to 47684 is C, the nucleotide at a positioncorresponding to 53095 is G, the nucleotide at a position correspondingto 56493 is T, the nucleotide at a position corresponding to 56586 is G,the nucleotide at a position corresponding to 56887 is C, the nucleotideat a position corresponding to 72076 is T, the nucleotide at a positioncorresponding to 74154 is C, and/or the sequence of AAG occurs betweennucleotides corresponding to positions 47669 and 47670.

[0141] Additionally, aspects of the present invention include the A2Mcoding sequences and cDNAs of SEQ ID NOs: 2-8 as modified by anucleotide(s) change at least one SNP and/or mutation selected from thegroup consisting of 12e, 14e, 20e, and 30e. More embodiments concern thenucleic acids of SEQ ID NOs: 1-8 having nucleotide(s) variations at oneor more previously described SNPs and/or mutations for A2M (e.g. SNPsand/or mutations provided in Table 2) in addition to a nucleotide(s)change at least one SNP and/or mutation selected from the groupconsisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i,21i, 28i and 30e.

[0142] In this regard, the nucleic acid embodiments described herein canhave from 9 to approximately 88,624 consecutive nucleotides so long asthe sequence contains nucleotide(s) variation at a SNP and/or mutationselected from the group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1,14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e, for example, or thenucleotides specified for the particular locations within SEQ ID NO: 1as set forth in (c) and (d) immediately above. Some of thesecompositions, for example, include nucleic acids having any numberbetween 9-50, 16-50, 17-50, 18-50, 19-50, 50-100, 100-500, 500-1000,1000-10,000, 10,000-50,000, or 50-88,634 consecutive nucleotides of SEQ.ID. NO. 1, wherein said nucleic acid contains a SNP and/or mutationselected from the group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1,14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e (e.g., greater than or equal to9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 125, 150, 175, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900,1000, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 5000,10,000, 25,000, 50,000, 75,000 and 88,624 consecutive nucleotides of asequence of SEQ ID NO:1 or portions of the above nucleotide list for SEQID NOs: 2-8, wherein said nucleic acid contains a nucleotide(s)variation at a SNP and/or mutation selected from the group consisting of6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and30e). In one embodiment, the nucleic acids comprise at least 12, 13, 14,15, 16, 17, 18, 19, 20 consecutive nucleotides of a sequence of SEQ IDNO:1 or SEQ ID NOs: 2-8, wherein said nucleic acid contains anucleotide(s) variation SNP and/or mutation selected from the groupconsisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i,21i, 28i and 30e, for example, or the nucleotides specified for theparticular locations within SEQ ID NO: 1 as set forth in (c) and (d)immediately above, or a complement thereof. In another embodiment, thenucleic acid embodiments comprise at least 20-30 consecutive nucleotidesof a sequence of SEQ ID NO: 1 or SEQ ID NOs: 2-8, wherein said nucleicacid contains a nucleotide(s) variation at a SNP and/or mutationselected from the group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1,14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e, for example, or thenucleotides specified for the particular locations within SEQ ID NO:1 asset forth in (c) and (d) immediately above, or complement thereof.

[0143] Several embodiments also include the above-described fragments ofthe nucleic acids of SEQ ID NOs: 1-8 having a nucleotide(s) variation atone or more previously described SNPs and/or mutations for A2M (e.g.SNPs and/or mutations provided in Table 2) in addition to anucleotide(s) variation at least one SNP and/or mutation selected fromthe group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1,20e, 20i, 21i, 28i and 30e, for example, the nucleotides specified forthe particular locations within SEQ ID NO:1 as set forth in (c) and (d)immediately above.

[0144] The nucleic acid embodiments described herein can also be alteredby mutation such as substitutions, additions, or deletions that providefor sequences encoding equivalent molecules. Due to the degeneracy ofnucleotide coding sequences, other DNA sequences that encodesubstantially the same polymorphic/mutant A2M amino acid sequence can bemade. These include, but are not limited to, nucleic acid sequencescomprising all or portions of SEQ ID NO: 1 or SEQ ID NOs: 2-8, whereinsaid nucleic acid sequences contain a a nucleotide(s) variation at a SNPand/or mutation selected from the group consisting of 6i, 12i.1, 12i.2,12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e, orcomplements thereof, which have been altered by the substitution ofdifferent codons that encode a functionally equivalent amino acidresidue within the sequence, thus producing a silent change.

[0145] The nucleic acid sequences described above have biotechnologicaland diagnostic use, e.g., in nucleic acid hybridization assays, Southernand Northern Blot analysis, etc. and the prognosis of neuropathies, suchas Alzheimer's Disease (AD). By using the nucleic acid sequencesdescribed herein, for example, probes that complement the polymorphicand/or mutant A2M genes or cDNAs can be designed and manufactured byoligonucleotide synthesis. Desirable probes comprise a nucleic acidsequence that is unique to the polymorphic and/or mutant A2M genes orcDNAs. These probes can be used to screen nucleic acids isolated fromtested individuals so as to identify the presence or absence of apolymorphism or combination of polymorphisms indicative of an altered,for example increased, risk of AD. Analysis can involve denaturinggradient gel electrophoresis or denaturing HPLC methods, for example.For guidance regarding probe design and denaturing gradient gelelectrophoresis or denaturing HPLC methods see, e.g., Ausubel et al.,1989, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y., including updated materials,U.S. Pat. Nos. 5,795,976; 5,585,236; 6,024,878; 6,210,885; Huber, etal., Chromatographia 37:653 (1993); Huber, et al., Anal. Biochem.212:351 (1993); Huber, et al., Anal. Chem. 67:578 (1995); O'Donovan etal., Genomics 52:44 (1998), Am J Hum Genet. Dec;67(6):1428-36 (2000);Ann Hum Genet. Sep:63 (Pt 5):383-91 (1999); Biotechniques,Apr;28(4):740-5 (2000); Biotechniques. Nov;29(5):1084-90, 1092 (2000);Clin Chem. Aug;45(8 Pt 1):1133-40 (1999); Clin Chem. Apr;47(4):635-44(2001); Genomics. Aug 15;52(1):44-9 (1998); Genomics. Mar15;56(3):247-53 (1999); Genet Test.;1(4):237-42 (1997-98); GenetTest.:4(2):125-9 (2000); Hum Genet. Jun;106(6):663-8 (2000); Hum Genet.Nov;107(5):483-7 (2000); Hum Genet. Nov;107(5):488-93 (2000); Hum Mutat.Dec;16(6):518-26 (2000); Hum Mutat. 15(6):556-64 (2000); Hum Mutat.Mar;17(3):210-9 (2001); J Biochem Biophys Methods. Nov 20;46(1-2):83-93(2000); J Biochem Biophys Methods. Jan 30;47(1-2):5-19 (2001); MutatRes. Nov 29;430(1): 13-21(1999); Nucleic Acids Res. Mar 1 ;28(5):E13(2000); and Nucleic Acids Res. Oct 15;28(20):E89 (2000), all of which,including the references contained therein, are hereby expresslyincorporated by reference in their entireties.

[0146] Also provided herein are oligonucleotides that can serve asprimers. Such oligonucleotides can be made, for example, by conventionaloligonucleotide synthesis for use in isolation and diagnostic proceduresthat employ the Polymerase Chain Reaction (PCR) or other enzyme-mediatednucleic acid amplification techniques or primer extension techniques.For a review of PCR technology, see Molecular Cloning to GeneticEngineering White, B. A. Ed. in Methods in Molecular Biology 67: HumanaPress, Totowa (1997), the disclosure of which is incorporated herein byreference in its entirety and the publication entitled “PCR Methods andApplications” (1991, Cold Spring Harbor Laboratory Press), thedisclosure of which is incorporated herein by reference in its entirety.

[0147] Oligonucleotide primers provided herein can contain a sequence ofnucleotides that specifically hybridizes adjacent to or at a polymorphicregion of the A2M gene spanning a nucleotide position corresponding toany of the following nucleotide positions of SEQ ID NO: 1: 37221, 45269,45088, 45125, 47519, 47684, 53095, 56493, 56586, 56887, 72076, 74154 and47669, or the complementary positions thereof adjacent to or at apolymorphic region of an A2M cDNA spanning a nucleotide positioncorresponding to any of the following positions: 1339, 1730, 2574 and3912 of SEQ ID NOs: 3 and 5; 1338, 1729, 2573 and 3911 of SEQ ID NO: 7;and 38 and 1376 of SEQ ID NO: 4. In particular embodiments, theoligonucleotides hybridize to a polymorphic region of the A2M gene underconditions of moderate or high stringency. Also provided areoligonucleotides, such as primers and probes that are the complements ofthese primers and probes. In particular embodiments, the probes orprimers contain a number of nucleotides sufficient to allow specifichybridization to the target nucleotide sequence. In particularembodiments of the probes and primers provided herein, the molecules areof sufficient length to specifically hybridize to portions of an A2Mgene at polymorphic sites. Typically such lengths depend upon thecomplexity of the source organism genome. For humans such lengthsgenerally are at least 14, 15, 16, 17, 18 or 19 nucleotides, andtypically may be at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400 or 500 or more nucleotides. In other embodiments, such lengths ofthe probes and primers provided are not more than 14, 15, 16, 17, 18 or19 nucleotides, and further may be not more than 20, 30, 40, 50, 60, 70,80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotidesin length.

[0148] For amplification of mRNAs, it is within the scope of theinvention to reverse transcribe mRNA into. cDNA followed by PCR(RT-PCR); or, to use a single enzyme for both steps as described in U.S.Pat. No. 5,322,770, the disclosure of which is incorporated herein byreference in its entirety. Another technique involves the use of ReverseTranscriptase Asymmetric Gap Ligase Chain Reaction (RT-AGLCR), asdescribed by Marshall R. L. et al. (PCR Methods and Applications4:80-84, 1994), the disclosure of which is incorporated herein byreference in its entirety. In each of these amplification procedures,primers on either side of the sequence to be amplified are added to asuitably prepared nucleic acid sample along with dNTPs and athermostable polymerase, such as Taq polymerase, Pfu polymerase, or Ventpolymerase. The nucleic acid in the sample is denatured and the primersare specifically hybridized to complementary nucleic acid sequences inthe sample. The hybridized primers are then extended. Thereafter,another cycle of denaturation, hybridization, and extension isinitiated. The cycles are repeated multiple times to produce anamplified fragment containing the nucleic acid sequence between theprimer sites. PCR has further been described in several patentsincluding U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188,. thedisclosure of which is incorporated herein by reference in theirentirety.

[0149] The primers are selected to be substantially complementary to aportion of the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NOs: 2-8that is downstream and upstream of the SNP and/or mutation to bedetected such that the fragment produced by the amplification orextension reaction contains the SNP and/or mutant. Preferably, primersare designed to be downstream and upstream of at least one of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e, forexample downstream or upstream of a nucleotide position corresponding toany of the following positions: 1339, 1730, 2574 and 3912 of SEQ ID NOs:3 and 5; 1338, 1729, 2573 and 3911 of SEQ ID NO: 7; and 38 and 1376 ofSEQ ID NO: 4, thereby allowing the sequences between the primers to beamplified or extended. Primers are desirably 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 and 30 nucleotides in length. The formationof stable hybrids depends on the melting temperature (Tm) of the DNA.The Tm depends on the length of the primer, the ionic strength of thesolution and the G+C content. The higher the G+C content of the primer,the higher is the melting temperature because G:C pairs are held bythree H bonds whereas A:T pairs have only two. The G+C content of theamplification primers of the present invention preferably ranges between10 and 75%, more preferably between 35 and 60%, and most preferablybetween 40 and 55%. The appropriate length for primers under aparticular set of assay conditions can be empirically determined by oneof skill in the art.

[0150] The spacing of the primers relates to the length of the segmentto be amplified. In the context of the present invention, amplifiedsegments carrying nucleotides corresponding to a nucleotide location of6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28iand/or 30e can range in size from at least about 25 bp to 35 kb.Amplification fragments that are any number from 25-1000 bp, 50-1000 bp,and fragments that are any number from 100-600 bp are common. It will beappreciated that amplification primers can be of any sequence thatallows for specific amplification of a region of a polymorphic and/ormutant A2M gene and can, for example, include modifications such asrestriction sites to facilitate cloning.

[0151] The PCR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequences of polymorphic and/or mutantA2M gene. The PCR fragment can then be used to isolate a full lengthcDNA clone by a variety of methods. For example, the amplified fragmentcan be labeled and used to screen a cDNA library, such as abacteriophage cDNA library. Alternatively, the labeled fragment can beused to isolate genomic clones via the screening of a genomic library.

[0152] Aspects of the invention also encompass (a) DNA vectors thatcontain any of the foregoing nucleic acid sequences; (b) DNA expressionvectors that contain any of the foregoing nucleic acid sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences; and (c) genetically engineered hostcells that contain any of the foregoing nucleic acid sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences in the host cell. These recombinantconstructs are capable of replicating autonomously in a host cell.Alternatively, the recombinant constructs can become integrated into thechromosomal DNA of a host cell.

[0153] As used herein, regulatory elements include, but are not limitedto, inducible and non-inducible promoters, enhancers, operators andother elements known to those skilled in the art that drive and regulateexpression. Such regulatory elements include, but are not limited to,the cytomegalovirus hCMV immediate early gene, the early or latepromoters of SV40 adenovirus, the lac system, the trp system, the TACsystem, the TRC system, the major operator and promoter regions of phageA, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase, the promoters of acid phosphatase, and thepromoters of the yeast α-mating factors.

[0154] In addition, recombinant polymorphic and/or mutant A2M-encodingnucleic acid sequences can be engineered so as to modify processing orexpression of the protein. For example, and not by way of limitation,the polymorphic and/or mutant A2M genes can be combined with a promotersequence and/or ribosome binding site, or a signal sequence can beinserted upstream of A2M-encoding sequences to permit secretion of theA2M protein and thereby facilitate harvesting or bioavailability.Additionally, a given polymorphic and/or mutant A2M nucleic acid can bemutated in vitro or in vivo, to create and/or destroy translation,initiation, and/or termination sequences, or to create variations incoding regions and/or form new restriction sites or destroy preexistingones, or to facilitate further in vitro modification. Any technique formutagenesis known in the art can be used, including but not limited to,in vitro site-directed mutagenesis. (Hutchinson et al., J. Biol. Chem.,253:6551 (1978), herein incorporated by reference).

[0155] Further, nucleic acids encoding other proteins or domains ofother proteins can be joined to nucleic acids encoding polymorphicand/or mutant A2M proteins or fragments thereof so as to create a fusionprotein. Nucleotides encoding fusion proteins can include, but are notlimited to, a full length polymorphic and/or mutant A2M protein, atruncated polymorphic and/or mutant A2M protein or a peptide fragment ofa polymorphic and/or mutant A2M protein fused to an unrelated protein orpeptide, such as for example, a transmembrane sequence, which anchorsthe A2M peptide fragment to the cell membrane; an Ig Fc domain whichincreases the stability and half life of the resulting fusion protein(e.g., A2M-Ig); or an enzyme, fluorescent protein, luminescent proteinwhich can be used as a marker (e.g., an A2M-Green Fluorescent Protein(“A2M-GFP”) fusion protein). The fusion proteins are useful asbiotechnological tools or pharmaceuticals or both, as will be discussedinfra. The section below describes several of the polypeptides of theinvention and methods of making these molecules.

[0156] The disclosed nucleic acids and others that can be obtained usingmethods described herein may be transferred into a host cell such asbacteria, yeast, insect, mammalian, or plant cell for recombinantexpression therein. Thus, provided herein are recombinant cellscontaining an A2M gene or a portion or portions thereof, such as, forexample, a transcriptional control region (including, for example, apromoter and 3′ untranslated (UTR) sequences) and/or a coding sequenceof an A2M gene. The A2M gene or portion(s) thereof contains at least onepolymorphic region and is thus referred to as a polymorphic A2M gene orportion(s) thereof. An “A2M gene or a portion or portions thereof”includes an A2M cDNA or portion(s) thereof

[0157] Cells containing nucleic acids encoding polymorphic A2M proteins,and vectors and cells containing the nucleic acids as provided hereinpermit production of the polymorphic proteins, as well as antibodies tothe proteins. This provides a means to prepare synthetic or recombinantpolymorphic proteins and fragments thereof that are substantially freeof contamination from other proteins, the presence of which caninterfere with analysis of the polymorphic proteins. In addition, thepolymorphic proteins may be expressed in combination with selected otherproteins that the protein of interest may associate with in cells. Theability to selectively express the polymorphic proteins alone or incombination with other selected proteins makes it possible to observethe functioning of the recombinant polymorphic proteins within theenvironment of a cell.

[0158] Recombinant cells provided herein may be used for numerouspurposes. For example, the cells may be used in testing polymorphic A2Mgenes or portion(s) thereof for characterization of phenotypic outcomescorrelated with the particular polymorphisms. The cells may also be usedin the production of recombinant A2M protein. Such protein may be used,for example, in assays for molecules that bind to, and in particularaffect the activity of, A2M. The proteins may also be used in theproduction of antibodies specific for the protein. Additionally, therecombinant A2M protein may be used as a source of a protease inhibitor.Recombinant cells containing polymorphic A2M genes or portion(s) thereofmay also be used in methods of identifying agents that modulate A2M geneand protein expression and/or activity or that modulate a biologicalevent characteristic of a disease or disorder involving altered A2M geneand/or protein expression or function which may be candidate treatmentsfor a disease or disorder.

[0159] Also provided herein are methods of producing recombinant cellsby introducing nucleic acid containing a polymorphic A2M gene orportion(s) as described herein thereof into a cell. The cell may be anytransfectable cell. Such cells, and methods of introducing heterologousnucleic acids into the cells, are known to those of skill in the art.

[0160] The exogenous nucleic acid containing a polymorphic A2M gene orportion(s) thereof that is used in the generation of recombinant cellsprovided herein contains, in particular embodiments, a sequence ofnucleotides that ultimately provides for a product upon transcription ofthe A2M gene or portion(s) thereof. The product can be, for instance,RNA and/or a protein translated from a transcript. For example, theproduct can be A2M mRNA and/or an A2M protein or a reporter moleculesuch as a reporter protein. If the polymorphic A2M gene or portion(s)thereof being used in the generation of recombinant cells providedherein does not contain sequences that provide for transciption of theA2M gene or portion(s) thereof, any appropriate transcription controlsequences, such as a promoter, from any appropriate source which willprovide for transciption of the A2M gene or portion(s) thereof in thecell can be used. If the polymorphism(s) occur in a transcriptioncontrol region of an A2M gene, the polymorphic control region of thegene can be isolated or synthesized and operatively linked to nucleicacid encoding a reporter molecule, e.g., galactosidase, a fluorescentprotein such as green fluorescent protein, or some other readilydetectable molecule, or nucleic acid encoding an A2M protein. Theresultant fusion gene can be used as the transgene that is introducedinto a host cell for use in development of recombinant cells therefrom.The patterns and levels of expression of the reporter or other moleculein the recombinant cells can be analyzed and compared to those in cellscontaining a fusion gene in which a wild-type or reference A2Mtranscription control region sequence is operatively linked to nucleicacid encoding a reporter or other molecule.

[0161] Polymorphic and/or mutant A2 M Polypeptides

[0162] Isolated or purified polymorphic and/or mutant A2M polypeptidesand fragments of these molecules at least 3 amino acids in length, whichcontain at least one of the mutations identified in Table 1, areembodiments of the invention. In some contexts, the term “polymorphicand/or mutant A2M polypeptides” refers not only to the full-lengthpolymorphic and/or mutant A2M proteins but also to fragments of thesemolecules at least 3 amino acids in length but containing at least oneof the mutations identified in Table 1.

[0163] The nucleic acids encoding the A2M polypeptides or fragmentsthereof, described in the previous section, can be manipulated usingconventional techniques in molecular biology so as to create recombinantconstructs that express polymorphic and/or mutant A2M polypeptides. Thepolymorphic and/or mutant A2M polypeptides or fragments thereof of theinvention, include but are not limited to, those containing as a primaryamino acid sequence all or part of the amino acid sequence encoded bySEQ ID NO: 1, SEQ ID NO: 2 (encoding SEQ ID NO: 9) or SEQ ID NOs: 3-8(encoding SEQ ID NOs: 10-15), as modified by a SNP and/or mutationdescribed in Table 1 (for example, 14e, 20e and 30e), and fragments ofthese proteins at least three amino acids in length but including atleast one of the mutations listed in Table 1, including alteredsequences in which functionally equivalent amino acid residues aresubstituted for residues within the sequence resulting in a silentchange. The A2M peptide fragments of the invention can be, for example,any number of between 4-20, 20-50, 50-100, 100-300, 300-600, 600-1000,1000-1450 consecutive amino acids of SEQ. ID NOs. 9-15 (e.g., less thanor equal to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 250, 300, 350, 400,500, 600, 700, 800, 900, 1000, and 1450 amino acids in length of SEQ IDNOs: 9-15). Polypeptides of the present invention also contemplate thepolypeptides of SEQ ID NOs: 9-15 or fragments thereof encoded by thenucleic acids of SEQ ID NOs: 2-8 having one or more previously describedSNPs and/or mutations for A2M which affect the A2M polypeptide (e.g.some SNPs and/or mutations provided in Table 2) in addition to at leastone SNP and/or mutation selected from the group consisting of 14e, 20eand 30e.

[0164] Embodiments also include isolated or purified polymorphic and/ormutant A2M polypeptides that have one or more amino acid residues withinthe polypeptide that are substituted by another amino acid of a similarpolarity that acts as a functional equivalent, resulting in a silentalteration. Substitutes for an amino acid within the sequence can beselected from other members of the class to which the amino acidbelongs. For example, the non-polar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine. The polar neutral amino acids includeglycine, serine, threonine, cysteine, tyrosine, asparagine andglutamine. The positively charged (basic) amino acids include arginine,lysine, and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. The aromatic amino acidsinclude phenylalanine, tryptophan, and tyrosine.

[0165] The sequences, constructs, vectors, clones, and other materialscomprising the embodiments of the present invention can be in enrichedor isolated form. As used herein, “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsfrom about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated.The term “isolated” requires that the material be removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide present ina living animal is not isolated, but the same polynucleotide, separatedfrom some or all of the coexisting materials in the natural system, isisolated. It is also advantageous that the sequences be in purifiedform. The term “purified” does not require absolute purity; rather, itis intended as a relative definition. Isolated proteins have beenconventionally purified to electrophoretic homogeneity by Coomassiestaining, for example. Purification of starting material or naturalmaterial to at least one order of magnitude, preferably two or threeorders, and more preferably four or five orders of magnitude isexpressly contemplated.

[0166] The polymorphic and/or mutant A2M polypeptides described hereincan be prepared by chemical synthesis methods (such as solid phasepeptide synthesis) using techniques known in the art such as those setforth by Merrifield et al., J. Am. Chem. Soc. 85:2149 (1964), Houghtenet al., Proc. Notl. Acad. Sci. USA, 82:51:32 (1985), Stewart and Young(Solid phase peptide synthesis, Pierce Chem Co., Rockford, Ill. (1984),and Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., all of which are hereby incorporated byreference in their entireties. Such polypeptides can be synthesized withor without a methionine on the amino terminus. Chemically synthesizedpolypeptides can be oxidized using methods set forth in these referencesto form disulfide bridges.

[0167] While the polymorphic and/or mutant A2M polypeptides andfragments thereof can be chemically synthesized, it can be moreeffective to produce these molecules by recombinant DNA technology usingtechniques well known in the art. Such methods can be used to constructexpression vectors containing the polymorphic and/or mutant A2Mnucleotide sequences, for example, and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. Alternatively, RNA capable of encoding anpolymorphic and/or mutant A2M polypeptide sequences and fragmentsthereof can be chemically synthesized using, for example, synthesizers.See, for example, the techniques described in Oligonucleotide Synthesis,1984, Gait, M. J. ed., IRL Press, Oxford, which is incorporated byreference herein in its entirety.

[0168] In several embodiments, polymorphic and/or mutant A2M nucleicacids and polypeptides are expressed in a cell line. For example, somecells are made to express the a polymorphic and/or mutant A2Mpolypeptide having the sequence encoded by SEQ ID NOs: 2-8 or suchnucleic acids having one or more previously described SNPs and/ormutations for A2M which affect the A2M polypeptide in addition to atleast one SNP and/or mutation selected from the group consisting of 14e,20e and 30e. A variety of host-expression vector systems can be utilizedto express the polymorphic and/or mutant A2M nucleic acids andpolypeptides of the invention. The expression systems that can be usedinclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli or B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing polymorphicand/or mutant A2M nucleotide sequences; yeast (e.g., Saccharomyces,Pichia) transformed with recombinant yeast expression vectors containingthe polymorphic and/or mutant A2M nucleotide sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,Baculovirus) containing the polymorphic and/or mutant A2M sequences;plant cell systems infected with recombinant virus expression vectors(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing polymorphic and/or mutant A2M nucleotide sequences;or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

[0169] In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for thepolymorphic and/or mutant A2M gene product being expressed. For example,when a large quantity of such a protein is to be produced, for thegeneration of pharmaceutical compositions of polymorphic and/or mutantA2M polypeptide or for raising antibodies to the polymorphic and/ormutant A2M polypeptide, for example, vectors which direct the expressionof high levels of fusion protein products that are readily purified canbe desirable. Such vectors include, but are not limited, to the E. coliexpression vector pUR278 (Ruther et al., EMBO J., 2:1791 (1983), inwhich the polymorphic and/or mutant A2M nucleic acids can be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res., 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.,264:5503-5509 (1989)); and the like, herein expressly incorporated byreference. pGEX vectors can also be used to express foreign polypeptidesas fusion proteins with glutathione S-transferase (GST). In general,such fusion proteins are soluble and can be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The PGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene product can be released from the GST moiety.

[0170] In an insect system, Autographa californica nuclear polyhedrosisvirus (ACNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The polymorphic and/or mutant A2Mnucleic acid sequences can be cloned individually into non-essentialregions (for example the polyhedrin gene) of the virus and placed undercontrol of an AcNPV promoter (for example the polyhedrin promoter).Successful insertion of polymorphic and/or mutant A2M nucleic acidsequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus, (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed. (E.g., see Smith et al., J. Virol. 46: 584(1983); and Smith, U.S. Pat. No. 4,215,051, all of which are herebyexpressly incorporated by reference in their entireties).

[0171] In mammalian host cells, a number of viral-based expressionsystems can be utilized. In cases where an adenovirus is used as anexpression vector, the polymorphic and/or mutant A2M nucleotide sequenceof interest can be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene can then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing the polymorphic and/ormutant A2M gene product in infected hosts. (E.g., See Logan & Shenk,Proc. Natl. Acad. Sci. USA 81:3655-3659 (1984), herein expresslyincorporated by reference in its entirety). Specific initiation signalscan also be required for efficient translation of inserted nucleotidesequences. These signals include the ATG initiation codon and adjacentsequences. In cases where an entire polymorphic and/or mutant A2M geneor cDNA, including its own initiation codon and adjacent sequences, isinserted into the appropriate expression vector, no additionaltranslational control signals are needed.

[0172] However, in cases where only a portion of the polymorphic and/ormutant A2M coding sequence is inserted, exogenous translational controlsignals, including, perhaps, the ATG initiation codon, may be provided.Furthermore, the initiation codon is desirably in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression can be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (SeeBittner et al., Methods in Enzymol, 153:516-544 (1987)).

[0173] In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products areimportant for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3, and W138.

[0174] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines that stablyexpress the polymorphic and/or mutant A2M sequences described herein canbe engineered. Rather than using expression vectors that contain viralorigins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA, engineered cells are allowed to grow for 1-2 days in anenriched media, and then are switched to a selective media. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci which in turn are cloned and expandedinto cell lines. This method is advantageously used to engineer celllines which express the polymorphic and/or mutant A2M gene product. Suchengineered cell lines are particularly useful in screening andevaluation of compounds that affect the endogenous activity of thepolymorphic and/or mutant A2M gene product.

[0175] A number of selection systems can be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler, et al.,Cell 11:223 (1977), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:2026 (1962), andadenine phosphoribosyltransferase (Lowy, et al., Cell 22:817 (1980)genes can be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., Proc. Natl. Acad. Sci. USA 77:3567 (1980); O'Hare, et al., Proc.Natl. Acad. Sci. USA 78:1527 (1981); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., J. Mol. Biol. 150:1 (1981); and hygro, whichconfers resistance to hygromycin (Santerre, et al., Gene 30:147 (1984)).

[0176] Alternatively, any fusion protein can be readily purified byutilizing an antibody specific for the fusion protein being expressed.For example, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines. (Janknecht, et al., Proc. Natl. Acad. Sci. USA 88: 8972-8976(1991)). In this system, the gene of interest is subcloned into aVaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni⁺ nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

[0177] The polymorphic and/or mutant A2M nucleic acids and polypeptidescan also be expressed in plants, insects, and animals so as to create atransgenic organism. Plants and insects of almost any species can bemade to express the polymorphic and/or mutant A2M nucleic acids and/orpolypeptides, described herein. Desirable transgenic plant systemshaving one or more of these sequences include Arabadopsis, Maize, andChlamydomonas. Desirable insect systems having one or more of thepolymorphic and/or mutant A2M nucleic acids and/or polypeptides include,for example, D. melanogaster and C. elegans. Animals of any species,including, but not limited to, amphibians, reptiles, birds, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, dogs, cats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees can be used togenerate polymorphic and/or mutant A2M containing transgenic animals.Transgenic organisms of the invention desirably exhibit germlinetransfer of polymorphic and/or mutant A2M nucleic acids andpolypeptides. Still other transgenic organisms of the invention exhibitcomplete knockouts or point mutations of one or more of the A2M genesdescribed herein.

[0178] Any technique known in the art is preferably used to introducethe polymorphic and/or mutant A2M transgene into animals to produce thefounder lines of transgenic animals or to knock out or replace existingA2M genes. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985); genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989); electroporation of embryos (Lo, Mol Cell. Biol. 3:1803-1814(1983); and sperm-mediated gene transfer (Lavitrano et al., Cell57:717-723 (1989); etc. For a review of such techniques, see Gordon,Transgenic Animals, Intl. Rev. Cytol. 115:171-229 (1989), which isincorporated by reference herein in its entirety.

[0179] Aspects of the invention also concern transgenic animals thatcarry a polymorphic and/or mutant A2M transgene in all their cells, aswell as animals that carry the transgene in some, but not all theircells, i.e., mosaic animals. The transgene can be integrated as a singletransgene or in concatamers, e.g., head-to-head tandems or head-to-tailtandems. The transgene can also be selectively introduced into andactivated in a particular cell type by following, for example, theteaching of Lasko et al. (Lasko, M. et al., Proc. Natl. Acad. Sci. USA89: 6232-6236 (1992), herein expressly incorporated by reference in itsentirety). The regulatory sequences required for such a cell-typespecific activation will depend upon the particular cell type ofinterest, and will be apparent to those of skill in the art.

[0180] When it is desired that the polymorphic and/or mutant A2M genetransgene be integrated into the chromosomal site of the endogenous A2Mgene, gene targeting is preferred. Briefly, when such a technique is tobe utilized, vectors containing some nucleotide sequences homologous tothe endogenous A2M gene are designed for the purpose of integrating, viahomologous recombination with chromosomal sequences, into and disruptingthe function of the nucleotide sequence of the endogenous A2M gene. Thetransgene can also be selectively introduced into a particular celltype, thus inactivating the endogenous A2M gene in only that cell type,by following, for example, the teaching of Gu et al. (Gu, et al.,Science 265: 103-106 (1994), herein expressly incorporated by referencein its entirety). The regulatory sequences required for such a cell-typespecific inactivation will depend upon the particular cell type ofinterest, and will be apparent to those of skill in the art.

[0181] Once transgenic animals have been generated, the expression ofthe recombinant A2M gene can be assayed utilizing standard techniques.Initial screening can be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals can also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. The section below describes antibodies of theinvention and methods of making these molecules.

[0182] Cells and transgenic animals containing nucleic acids thatinclude variant A2M gene or cDNA sequences as described herein havenumerous uses. For example, such cells and animals can be used inmethods of assessing candidate agents that modulate A2M activity and/orexpression, and candidate therapeutic agents for the treatment ofdiseases, such as neurodegenerative diseases, e.g., AD. Such cells andanimals can also be used to assess the effects of a particular variantof a polymorphism. For example, transgenic animals in which nucleic acidcontaining a particular variant of a polymorphism has been introducedmay be analyzed for a particular phenotype. The transgenic animal may beone in which the wild-type gene or predominant allele may have beenknocked out. RNA and/or protein is compared in the transgenic animalharboring the allelic variant with an animal harboring a differentallele, e.g., a predominant or reference allele. For example, thevariant may result in alterations of RNA levels or RNA stability or inincreased or decreased synthesis of the associated protein and/oraberrant tissue distribution or intracellular localization of theassociated protein, altered phosphorylation, glycosylation and/oraltered activity of the protein. Furthermore, various molecular,cellular and organismal manifestations of a disease can be monitored.For example, to assess a polymorphism for an effect that may be relatedto Alzheimer's disease, certain characteristic features of the disease,such as APP gene products, particularly A protein, neurite plaques,deficits of memory and learning and neurodegeneration of specificsystems of cells may be evaluated in a transgenic animal containingnucleic acid containing the polymorphism. Such analysis could also beperformed in cultured cells into which the variant allele gene orportion thereof is introduced. If the host cell contains a differentallele of the same gene, it is possible to replace the endogenous genewith the variant gene in the cell, if desired. These effects can bedetermined according to methods known in the art and as described below.Particular variants of a polymorphism can be assayed individually or incombination.

[0183] Antibodies Specific for Polymorphic and/or mutant A2MPolypeptides

[0184] Following synthesis or expression and isolation or purificationof the A2M protein or a portion thereof, the isolated or purifiedprotein can be used to generate antibodies and tools for identifyingagents that interact with polymorphic and/or mutant A2M polypeptides.Depending on the context, the term “antibodies” can encompasspolyclonal, monoclonal, chimeric, single chain, Fab fragments andfragments produced by a Fab expression library. Antibodies thatrecognize polymorphic and/or mutant A2M polypeptides have many usesincluding, but not limited to, biotechnological applications,therapeutic/prophylactic applications, and diagnostic applications.

[0185] For the production of antibodies, various hosts including goats,rabbits, rats, mice, etc. can be immunized by injection with polymorphicand/or mutant A2M polypeptides, in particular, any portion, fragment oroligopeptide that retains immunogenic properties. Depending on the hostspecies, various adjuvants can be used to increase immunologicalresponse. Such adjuvants include, but are not limited to, Freund's,mineral gels such as aluminum hydroxide, and surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (BacillusCalmette-Guerin) and Corynebacterium parvum are also potentially usefuladjuvants.

[0186] Peptides used to induce specific antibodies can have an aminoacid sequence consisting of at least three amino acids, and preferablyat least 10 to 15 amino acids. Preferably, short stretches of aminoacids encoding fragments of polymorphic and/or mutant A2M polypeptidescontaining one or more of the mutations described in Table 1 are fusedwith those of another protein such as keyhole limpet hemocyanin suchthat an antibody is produced against the chimeric molecule. Whileantibodies capable of specifically recognizing polymorphic and/or mutantA2M polypeptides can be generated by injecting synthetic 3-mer, 10-mer,and 15-mer peptides that correspond to a protein sequence of polymorphicand/or mutant A2M polypeptides into mice, a more diverse set ofantibodies can be generated by using recombinant polymorphic and/ormutant A2M polypeptides.

[0187] To generate antibodies to polymorphic and/or mutant A2Mpolypeptides, substantially pure polypeptides are isolated from atransfected or transformed cell. The concentration of the polypeptide inthe final preparation is adjusted, for example, by concentration on anAmicon filter device, to the level of a few micrograms/ml. Monoclonal orpolyclonal antibody to the polypeptide of interest can then be preparedas follows:

[0188] Monoclonal antibodies to polymorphic and/or mutant A2Mpolypeptides can be prepared using any technique that provides for theproduction of antibody molecules by continuous cell lines in culture.These include, but are not limited to, the hybridoma techniqueoriginally described by Koehler and Milstein (Nature 256:495-497 (1975),the human B-cell hybridoma technique (Kosbor et al. Immunol Today 4:72(1983); Cote et al Proc Natl Acad Sci 80:2026-2030 (1983), and theEBV-hybridoma technique Cole et al. Monoclonal Antibodies and CancerTherapy, Alan R. Liss Inc, New York N.Y., pp 77-96 (1985), all of whichare hereby incorporated by reference in their entireties. In addition,techniques developed for the production of “chimeric antibodies”, thesplicing of mouse antibody genes to human antibody genes to obtain amolecule with appropriate antigen specificity and biological activitycan be used. (Morrison et al. Proc Natl Acad Sci 81:6851-6855 (1984);Neuberger et al. Nature 312:604-608(1984); Takeda et al. Nature314:452-454(1985), all of which are hereby incorporated by reference intheir entireties. Alternatively, techniques described for the productionof single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce specific single chain antibodies, hereby incorporated byreference. Antibodies can also be produced by inducing in vivoproduction in the lymphocyte population or by screening recombinantimmunoglobulin libraries or panels of highly specific binding reagentsas disclosed in Orlandi et al., Proc Natl Acad Sci 86: 3833-3837 (1989),and Winter G. and Milstein C; Nature 349:293-299 (1991), all of whichare hereby incorporated by reference in their entireties.

[0189] Antibody fragments that contain specific binding sites forpolymorphic and/or mutant A2M polypeptides can also be generated. Forexample, such fragments include, but are not limited to, the F(ab′)₂fragments that can be produced by pepsin digestion of the antibodymolecule and the Fab fragments that can be generated by reducing thedisulfide bridges of the F(ab′)₂ fragments. Alternatively, Fabexpression libraries can be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.(Huse W. D. et al. Science 256:1275-1281 (1989)).

[0190] By one approach, monoclonal antibodies to polymorphic and/ormutant A2M polypeptides are made as follows. Briefly, a mouse isrepetitively inoculated with a few micrograms of the selected protein orpeptides derived therefrom over a period of a few weeks. The mouse isthen sacrificed, and the antibody producing cells of the spleenisolated. The spleen cells are fused in the presence of polyethyleneglycol with mouse myeloma cells, and the excess unfused cells destroyedby growth of the system on selective media comprising aminopterin (HATmedia). The successfully fused cells are diluted and aliquots of thedilution placed in wells of a microtiter plate where growth of theculture is continued. Antibody-producing clones are identified bydetection of antibody in the supernatant fluid of the wells byimmunoassay procedures, such as ELISA, as originally described byEngvall, E., Meth. Enzymol. 70:419 (1980), and derivative methodsthereof. Selected positive clones can be expanded and their monoclonalantibody product harvested for use. Detailed procedures for monoclonalantibody production are described in Davis, L. et al. Basic Methods inMolecular Biology Elsevier, New York. Section 21-2, herein expresslyincorporated by reference in its entirety.

[0191] Polyclonal antiserum containing antibodies to heterogenousepitopes of a single protein can be prepared by immunizing suitableanimals with the expressed protein or peptides derived therefromdescribed above, which can be unmodified or modified to enhanceimmunogenicity. Effective polyclonal antibody production is affected bymany factors related both to the antigen and the host species. Forexample, small molecules tend to be less immunogenic than others and canrequire the use of carriers and adjuvant. Also, host animals vary inresponse to site of inoculations and dose, with both inadequate orexcessive doses of antigen resulting in low titer antisera. Small doses(ng level) of antigen administered at multiple intradermal sites appearsto be most reliable. An effective immunization protocol for rabbits canbe found in Vaitukaitis, J. et al. J. Clin. Endocrinol. Metab.33:988-991 (1971),herein expressly incorporated by reference in itsentirety.

[0192] Booster injections can be given at regular intervals, andantiserum harvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony, 0. et al., Chap. 19 in: Handbook of ExperimentalImmunology D. Wier (ed) Blackwell (1973). Plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12μM). Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher, D.,Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman,Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980). Antibodypreparations prepared according to either protocol are useful inquantitative immunoassays that determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively (e.g., in diagnostic embodimentsthat identify the presence of polymorphic and/or mutant A2M polypeptidesin biological samples). In the discussion that follows, several methodsof molecular modeling and rational drug design are described. Thesetechniques can be applied to identify molecules that interact withpolymorphic and/or mutant A2M polypeptides and, thereby modulate theirfunction.

[0193] Diagnostic Embodiments

[0194] Generally, the diagnostics of the invention can be classifiedaccording to whether the embodiment is a nucleic acid or protein-basedassay. Some diagnostic assays detect mutations or polymorphisms in A2Mnucleic acids or A2M proteins, which contribute to or place individualsat risk of acquiring neuropathies, such as AD. Other diagnostic assaysidentify and distinguish defects in A2M activities by detecting a levelof polymorphic and/or mutant A2M RNA or A2M protein in a tested subjectthat resembles the level of polymorphic and/or mutant A2M RNA or A2Mprotein in a subject suffering from a neuropathy (e.g., AD) or bydetecting a level of RNA or protein in a tested subject that isdifferent than a subject not suffering from a disease.

[0195] Additionally, the manufacture of kits that incorporate thereagents and methods described in the following embodiments so as toallow for the rapid detection and identification of individuals at riskof acquiring a neuropathy, such as AD, are contemplated. The diagnostickits can include a nucleic acid probe or an antibody or combinationsthereof, which specifically detect a polymorphic and/or mutant A2Mpolypeptide or nucleic acid or a nucleic acid probe or an antibody orcombinations thereof, which can be used to determine the level of RNA orprotein expression of one or more polymorphic and/or mutant A2M nucleicacids or polypeptides. The detection component of these kits willtypically be supplied in combination with one or more of the followingreagents. A support capable of absorbing or otherwise binding DNA, RNA,or protein will often be supplied. Available supports include membranesof nitrocellulose, nylon or derivatized nylon that can be characterizedby bearing an array of positively charged substituents. One or morerestriction enzymes, control reagents, buffers, amplification enzymes,and non-human polynucleotides like calf-thymus or salmon-sperm DNA canbe supplied in these kits.

[0196] Useful nucleic acid-based diagnostic techniques include, but arenot limited to, direct DNA sequencing, Southern Blot analysis,single-stranded confirmation analysis (SSCA), RNAse protection assay,dot blot analysis, nucleic acid amplification, and combinations of theseapproaches. The starting point for these analysis is isolated orpurified nucleic acid from a biological sample. If the diagnostic assayis designed to determine the presence of a polymorphic and/or mutant A2Mnucleic acid, any source of DNA including, but not limited to hair,cheek cells and blood can be used as a biological sample. The nucleicacid is extracted from the sample and can be amplified by a DNAamplification technique such as the Polymerase Chain Reaction (PCR)using primers that correspond to regions flanking DNA recognized as aSNP and/or mutation in the A2M gene (See Table 1).

[0197] Once a sufficient amount of DNA is obtained from an individual tobe tested, several methods can be used to detect a polymorphism and/ormutation. Direct DNA sequencing, either manual sequencing or automatedfluorescent sequencing can detect such sequence variations. Anotherapproach is the single-stranded confirmation polymorphism assay (SSCA)(Orita et al., Proc. Natl. Acad. Sci. USA 86:2776-2770 (1989), hereinincorporated by reference). This method, however, does not detect allsequence changes, especially if the DNA fragment size is greater than200 base pairs, but can be optimized to detect most DNA sequencevariation.

[0198] The reduced detection sensitivity is a disadvantage, but theincreased throughput possible with SSCA makes it an attractive, variablealternative to direct sequencing for mutation detection. The fragmentsthat have shifted mobility on SSCA gels are then sequenced to determinethe exact nature of the DNA sequence variation. Other approaches basedon the detection of mismatches between the two complimentary DNA strandsinclude clamped denaturing gel electrophoresis (CDGE) (Sheffield et al.,Am. J. Hum. Genet. 49:699-706 (1991)), heteroduplex analysis (HA) (Whiteet al., Genomics 12:301-306 (1992)), and chemical mismatch cleavage(CMC) (Grompe et al., Proc. Natl. Acad. Sci. USA 86:5855-5892 (1989),all of which, including the references contained therein, are herebyexpressly incorporated by reference in their entireties). A review ofcurrently available methods of detecting DNA sequence variation can befound in Grompe, Nature Genetics 5:111-117 (1993).

[0199] Seven well-known nucleic acid-based methods for confirming thepresence of a polymorphism are described below. Provided for exemplarypurposes only and not intended to limit any aspect of the invention,these methods include:

[0200] (1) single-stranded confirmation analysis (SSCA) (Orita et al.);

[0201] (2) denaturing gradient gel electrophoresis (DGGE) (Wartell etal., Nucl. Acids Res. 18:2699-2705 (1990) and Sheffield et al., Proc.Natl. Acad. Sci. USA 86:232-236 (1989)), both references hereinincorporated by reference;

[0202] (3) RNAse protection assays (Finkelstein et al., Genomics7:167-172 (1990) and Kinszler et al., Science 251:1366-1370 (1991)) bothreferences herein incorporated by reference;

[0203] (4) the use of proteins which recognize nucleotide mismatches,such as the E. Coli mutS protein (Modrich, Ann. Rev. Genet. 25:229-253(1991), herein incorporated by reference;

[0204] (5) allele-specific PCR (Rano and Kidd, Nucl. Acids Res. 17:8392(1989), herein incorporated by reference), which involves the use ofprimers that hybridize at their 3′ ends to a polymorphism and, if thepolymorphism is not present, an amplification product is not observed;and

[0205] (6) Amplification Refractory Mutation System (ARMS), as disclosedin European Patent Application Publication No. 0332435 and in Newton etal., Nucl. Acids Res. 17:2503-2516 (1989), both references hereinincorporated by reference; and

[0206] (7) temporal temperature gradient gel electrophoresis (TTGE), asdescribed by Bio-Rad in U.S./E.G. Bulletin 2103, herein incorporated byreference.

[0207] In SSCA, DGGE, TTGE, and RNAse protection assay, a newelectrophoretic band appears when the polymorphism is present. SSCA andTTGE detect a band that migrates differentially because the sequencechange causes a difference in single-strand, intramolecular basepairing, which is detectable electrophoretically. RNAse protectioninvolves cleavage of the mutant polynucleotide into two or more smallerfragments. DGGE detects differences in migration rates of sequencesusing a denaturing gradient gel. In an allele-specific oligonucleotideassay (ASOs) (Conner et al., Proc. Natl. Acad. Sci. USA 80:278-282(1983)), an oligonucleotide is designed that detects a specificsequence, and an assay is performed by detecting the presence or absenceof a hybridization signal. In the mutS assay, the protein binds only tosequences that contain a nucleotide mismatch in a heteroduplex betweenpolymorphic and non-polymorphic sequences. Mismatches, in this sense ofthe word refers to hybridized nucleic acid duplexes in which the twostrands are not 100% complementary. The lack of total homology resultsfrom the presence of one or more polymorphisms in an amplicon obtainedfrom a biological sample, for example, that has been hybridized to anon-polymorphic strand. Mismatched detection can be used to detect pointmutations in DNA or in an mRNA. While these techniques are lesssensitive than sequencing, they are easily performed on a large numberof biological samples and are amenable to array technology.

[0208] In some embodiments, nucleic acid probes that differentiatepolynucleotides encoding wild type A2M from polymorphic and/or mutantA2M are attached to a support in an ordered array, wherein the nucleicacid probes are attached to distinct regions of the support that do notoverlap with each other. Preferably, such an ordered array is designedto be “addressable” where the distinct locations of the probe arerecorded and can be accessed as part of an assay procedure. These probesare joined to a support in different known locations. The knowledge ofthe precise location of each nucleic acid probe makes these“addressable” arrays particularly useful in binding assays. The nucleicacids from a preparation of several biological samples are then labeledby conventional approaches (e.g., radioactivity or fluorescence) and thelabeled samples are applied to the array under conditions that permithybridization.

[0209] If a nucleic acid in the samples hybridizes to a probe on thearray, then a signal will be detected at a position on the support thatcorresponds to the location of the hybrid. Since the identity of eachlabeled sample is known and the region of the support on which thelabeled sample was applied is known, an identification of the presenceof the polymorphic variant can be rapidly determined. These approachesare easily automated using technology known to those of skill in the artof high throughput diagnostic or detection analysis.

[0210] Additionally, an opposite approach to that presented above can beemployed. Nucleic acids present in biological samples can be disposed ona support so as to create an addressable array. Preferably, the samplesare disposed on the support at known positions that do not overlap. Thepresence of nucleic acids having a desired polymorphism in each sampleis determined by applying labeled nucleic acid probes that complementnucleic acids that encode the polymorphism and detecting the presence ofa signal at locations on the array that correspond to the positions atwhich the biological samples were disposed. Because the identity of thebiological sample and its position on the array is known, theidentification of the polymorphic variant can be rapidly determined.These approaches are also easily automated using technology known tothose of skill in the art of high throughput diagnostic analysis.

[0211] Any addressable array technology known in the art can be employedwith this aspect of the invention. One particular embodiment ofpolynucleotide arrays is known as Genechips™, and has been generallydescribed in U.S. Pat. No. 5,143,854; PCT publications WO 90/15070 and92/10092. These arrays are generally produced using mechanical synthesismethods or light directed synthesis methods, which incorporate acombination of photolithographic methods and solid phase oligonucleotidesynthesis. (Fodor et al., Science, 251:767-777, (1991)). Theimmobilization of arrays of oligonucleotides on solid supports has beenrendered possible by the development of a technology generallyidentified as “Very Large Scale Immobilized Polymer Synthesis” (VLSPIS™)in which, typically, probes are immobilized in a high density array on asolid surface of a chip. Examples of VLSPIS™ technologies are providedin U.S. Pat. Nos. 5,143,854 and 5,412,087 and in PCT Publications WO90/15070, WO 92/10092 and WO 95/11995, which describe methods forforming oligonucleotide arrays through techniques such as light-directedsynthesis techniques. In designing strategies aimed at providing arraysof nucleotides immobilized on solid supports, further presentationstrategies were developed to order and display the oligonucleotidearrays on the chips in an attempt to maximize hybridization patterns anddiagnostic information. Examples of such presentation strategies aredisclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212, andWO 97/31256, all of which are hereby incorporated by reference in theirentireties.

[0212] A wide variety of labels and conjugation techniques are known bythose skilled in the art and can be used in various nucleic acid assays.There are several ways to produce labeled nucleic acids forhybridization or PCR including, but not limited to, oligolabeling, nicktranslation, end-labeling, or PCR amplification using a labelednucleotide. Alternatively, a nucleic acid encoding a polymorphic and/ormutant A2M polypeptide can be cloned into a vector for the production ofan mRNA probe. Such vectors are known in the art, are commerciallyavailable, and can be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3 or SP6 and labelednucleotides. A number of companies such as Pharmacia Biotech (PiscatawayN.J.), Promega (Madison Wis.), and U.S. Biochemical Corp (ClevelandOhio) supply commercial kits and protocols for these procedures.Suitable reporter molecules or labels include those radionuclides,enzymes, fluorescent, chemiluminescent, or chromogenic agents, as wellas, substrates, cofactors, inhibitors, magnetic particles and the like.

[0213] The RNAse protection method, briefly described above, is anexample of a mismatch cleavage technique that is amenable to arraytechnology. Preferably, the method involves the use of a labeledriboprobe that is complementary to polymorphic and/or mutant A2M nucleicacid sequences selected from the group consisting of 6i, 12i.1, 12i.2,12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e. The riboprobeand either mRNA or DNA isolated and amplified from a biological sampleare annealed (hybridized) and subsequently digested with the enzymeRNAse A, which is able to detect mismatches in a duplex RNAse structure.If a mismatch is detected by RNAse A, the polymorphic variant is notpresent in the sample and the enzyme cleaves at the site of the mismatchand destroys the riboprobe. Thus, when the annealed RNA is separated ona electrophoretic gel matrix, if a mismatch has been detected andcleaved by RNAse A, an RNA product will be seen which is much smallerthan the full length duplex RNA for the riboprobe and the mRNA or DNA.

[0214] Complements to the riboprobe can also be dispersed on an arrayand stringently probed with the products from the Rnase A digestionafter denaturing any remaining hybrids. In this case, if a mismatch isdetected and probe destroyed by Rnase A, the complements on the arraywill not anneal with the degraded RNA under stringent conditions. In asimilar fashion, DNA probes can be used to detect mismatches, throughenzymatic or chemical cleavage. See, e.g., Cotton, et al., Proc. Natl.Acad. Sci. USA 85:4397 (1988); Shenk et al., Proc. Natl. Acad. Sci. USA72:989 (1975); and Novack et al., Proc. Natl. Acad. Sci. USA 83:586(1986). Mismatches can also be detected by shifts in the electrophoreticability of mismatched duplexes relative to matched duplexes. (See, e.g.,Cariello, Human Genetics 42:726 (1988), herein incorporated byreference). With any of the techniques described above, the mRNA or DNAfrom a tested organism that corresponds to regions of an A2M gene havinga polymorphism selected from the group consisting of 6i, 12i.1, 12i.2,12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e can beamplified by PCR before hybridization.

[0215] The presence of polymorphic and/or mutant A2M polypeptides in aprotein sample can also be detected by using conventional assays. Forexample, antibodies immunoreactive with a polymorphic and/or mutant A2Mpolypeptide can be used to screen patient biological samples todetermine if said patients are at risk of acquiring AD or have apredilection to acquire AD. Additionally, antibodies that differentiatethe wild type A2M from polymorphic and/or mutant A2M polypeptides can beused to determine that an organism does not have a risk of acquiring ADor a predilection to acquire AD.

[0216] In preferred embodiments, antibodies are used toimmunoprecipitate the polymorphic and/or mutant A2M polypeptides fromsolution or are used to react with the polymorphic and/or mutant A2Mpolypeptides on Western or Immunoblots. Favored diagnostic embodimentsalso include enzyme-linked immunosorbant assays (ELISA),radioimmunoassays (RIA), immunoradiometric assays (IRMA) andimmunoenzymatic assays (IEMA), including sandwich assays usingmonoclonal and/or polyclonal antibodies. Exemplary sandwich assays aredescribed by David et al., in U.S. Pat. Nos. 4,376,110 and 4,486,530,hereby incorporated by reference. Other embodiments employ aspects ofthe immune-strip technology disclosed in U.S. Pat. Nos. 5,290,678;5,604,105; 5,710,008; 5,744,358; and 5,747,274, herein incorporated byreference.

[0217] In another preferred protein-based diagnostic, antibodies of theinvention are attached to a support in an ordered array wherein aplurality of antibodies are attached to distinct regions of the supportthat do not overlap with each other. As with the nucleic acid-basedarrays, the protein-based arrays are ordered arrays that are designed tobe “addressable” such that the distinct locations are recorded and canbe accessed as part of an assay procedure. These probes are joined to asupport in different known locations. The knowledge of the preciselocation of each probe makes these “addressable” arrays particularlyuseful in binding assays. For example, an addressable array can comprisea support having several regions to which are joined a plurality ofantibody probes that specifically recognize a particular A2M anddifferentiate the polymorphic and/or mutant A2M polypeptides from wildtype A2M.

[0218] Proteins are obtained from biological samples and are labeled byconventional approaches (e.g., radioactivity, calorimetrically, orfluorescently). The labeled samples are then applied to the array underconditions that permit binding. If a protein in the sample binds to anantibody probe on the array, then a signal will be detected at aposition on the support that corresponds to the location of theantibody-protein complex. Since the identity of each labeled sample isknown and the region of the support on which the labeled sample wasapplied is known, an identification of the presence, concentration,and/or expression level can be rapidly determined. That is, by employinglabeled standards of a known concentration of polymorphic and/or mutantA2M polypeptide or wild-type A2M, an investigator can accuratelydetermine the protein concentration of the particular A2M in a testedsample and can also assess the expression level of the A2M. Conventionalmethods in densitometry can also be used to more accurately determinethe concentration or expression level of the A2M. These approaches areeasily automated using technology known to those of skill in the art ofhigh throughput diagnostic analysis.

[0219] In another embodiment, an opposite approach to that presentedabove can be employed. Proteins present in biological samples can bedisposed on a support so as to create an addressable array. Preferably,the protein samples are disposed on the support at known positions thatdo not overlap. The presence of a protein encoding a polymorphic and/ormutant A2M polypeptide in each sample is then determined by applyinglabeled antibody probes that recognize epitopes specific for thepolymorphic and/or mutant A2M polypeptide. Because the identity of thebiological sample and its position on the array is known, anidentification of the presence, concentration, and/or expression levelof a particular polymorphism can be rapidly determined.

[0220] That is, by employing labeled standards of a known concentrationof polymorphic and/or mutant A2M polypeptides, an investigator canaccurately determine the concentration of A2M in a sample and from thisinformation can assess the expression level of the particular form ofA2M. Conventional methods in densitometry can also be used to moreaccurately determine the concentration or expression level of the A2M.These approaches are also easily automated using technology known tothose of skill in the art of high throughput diagnostic analysis. Asdetailed above, any addressable array technology known in the art can beemployed with this aspect of the invention and display the proteinarrays on the chips in an attempt to maximize antibody binding patternsand diagnostic information.

[0221] As discussed above, the presence or detection of one or more ofthe mutations and/or polymorphisms provided in Table 1 can provide adiagnosis that the tested subject is at risk of acquiring AD or has apredilection to acquire AD. Additional embodiments include thepreparation of diagnostic kits comprising detection components, such asantibodies, specific for one or more of the particular polymorphicvariants of A2M or A2M described herein. The detection component willtypically be supplied in combination with one or more of the followingreagents. A support capable of absorbing or otherwise binding RNA orprotein will often be supplied. Available supports for this purposeinclude, but are not limited to, membranes of nitrocellulose, nylon orderivatized nylon that can be characterized by bearing an array ofpositively charged substituents, and Genechips™ or their equivalents.One or more enzymes, such as Reverse Transcriptase and/or Taqpolymerase, can be furnished in the kit, as can dNTPs, buffers, ornon-human polynucleotides like calf-thymus or salmon-sperm DNA. Resultsfrom the kit assays can be interpreted by a healthcare provider or adiagnostic laboratory. Alternatively, diagnostic kits are manufacturedand sold to private individuals for self-diagnosis.

[0222] In addition to diagnosing disease according to the presence orabsence of a polymorphic and/or mutant A2M nucleic acid or A2Mpolypeptide, some diseases may result from skewed levels of wild-typeA2M as compared to polymorphic and/or mutant A2M. By monitoring thelevel of expression of specific A2M polypeptides, for example, adiagnosis can be made or a disease state can be identified. Similarly,by determining ratios of the level of expression of various A2Mpolypeptides a prognosis of health or disease can be made. The levels ofexpression of different types of A2M in various healthy individuals, aswell as, individuals suffering from AD can be determined, for example.These values can be recorded in a database and can be compared to valuesobtained from tested individuals. Additionally, the ratios or patternsof expression of various A2M polypeptides from both healthy and diseasedindividuals is recorded in a database. These analyses are referred to as“disease state profiles” and by comparing one disease state profile(e.g. from a healthy or diseased individual) to a disease state profilefrom a tested individual, a clinician can rapidly diagnose the presenceor absence of disease. .

[0223] The nucleic acid and protein-based diagnostic techniquesdescribed above can be used to detect the level or amount or ratio ofexpression of a particular A2M RNAs or A2M proteins in a tissue. Throughquantitative Northern hybridizations, In situ analysis,immunohistochemistry, ELISA, genechip array technology, PCR, and Westernblots, for example, the amount or level of expression of RNA or proteinfor a particular A2M (wild-type or mutant) can be rapidly determined andfrom this information ratios of A2M expression can be ascertained.Preferably, the expression levels of A2M genes having one or more of apolymorphism and/or mutation selected from the group consisting of 6i,12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30eare measured to determine the ratios.

[0224] Once the levels of various A2M polypeptides or nucleic acids aredetermined, the information can be recorded onto a computer readablemedia, such as a hard drive, floppy disk, DVD drive, zip drive, etc.After recording and the generation of a database comprising the levelsof expression of the various A2M polypeptides or nucleic acids studied,a comparing program is used which compares the levels of expression ofthe various A2M polypeptides or nucleic acids so as to create a ratio ofexpression. The following section describes the preparation ofpharmaceuticals having polymorphic and/or mutant A2M polypeptides orbinding partners, which can be administered to organisms in need tomodulate A2M activities.

[0225] Pharmacogenomics

[0226] It is likely that subjects having one or more different allelicvariants of the A2M gene will respond differently to drugs to treatassociated diseases or disorders. For example, alleles of the A2M genethat associate with neurodegenerative disease will be useful alone or inconjunction with other genes associated with the development ofneurodegenerative disease (e.g., APOE4) to predict a subject's response,either positive or negative, to a therapeutic drug. Multiplex primerextension assays or microarrays comprising probes for specific allelesare useful formats for determining drug response. A correlation betweendrug responses and specific alleles or combinations of alleles(haplotypes) of the A2M gene and other genes that associate with diseasecan be shown, for example, by clinical studies wherein the response,either positive or negative, to specific drugs of subjects havingdifferent allelic variants of polymorphic regions of the A2M gene aloneor in combination with allelic variants of other genes are compared.Such studies can also be performed using animal models, such as micehaving various alleles and in which, e.g., the endogenous uPA gene hasbeen inactivated such as by a knock-out mutation. Test drugs are thenadministered to the mice having different alleles and the response ofthe different mice to a specific compound is compared. Accordingly,assays, microarrays and kits are provided for determining the drug whichwill be best suited for treating a specific disease or condition in asubject based on the individual's genotype. For example, it will bepossible to select drugs which will be devoid of toxicity, or have thelowest level of toxicity possible for treating a subject having adisease or condition, e.g., neurodegenerative disease or Alzheimer'sdisease.

[0227] For example, therapeutic agents for treatment ofneurodegenerative disease that can be genetically profiled include, butare not limited to, ALCAR, Alpha-tocopherol (Vitamin E),), Ampalex,AN-1792 (AIP-001), Cerebrolysin, Daposone, Donepezil (Aricept), ENA-713(Exelon), Estrogen replacement therapy, Galanthamine (Reminyl), GinkgoBiloba extract, Huperzine A, Ibuprofen, Lipitor, Naproxen, Nefiracetam,Neotrofin, Memantine, Phenserine, Rofecoxib, Selegiline (Eldepryl),Tacrine (Cognex), Xanomeline (skin patch), Resperidone (Risperidol™),Neuroleptics, Benzodiazepenes, Valproate, Serotonin reuptake inhibitors(SRIs), Beta and Gamma Secretase Inhibitors, CX-516 (Ampalex), Statinsand AF-102B (Evoxac).

[0228] Other therapeutic agents for treatment of neurodegenerativedisease include those that are neuroprotective. Drugs withanti-oxidative properties, e.g., flupirtine, N-acetylcysteine,idebenone, melatonin, and also novel dopamine agonists (ropinirole andpramipexole) have been shown to protect neuronal cells from apoptosisand thus have been suggested for treating neurodegenerative disorderslike AD or PD. Also, free radical scavengers, calcium channel blockersand modulators of certain signal transduction pathways that mightprotect neurons from downstream effects of the accumulation of A-Betaintracellularly and/or extracellularly. Also, other agents likenon-steroidal anti-inflammatory drugs (NSAIDs) partly inhibitcyclooxygenase (COX) expression, as well as having a positive influenceon the clinical expression of AD. Distinct cytokines, growth factors andrelated drug candidates, e.g., nerve growth factor (NGF), or members ofthe transforming growth factor-beta (TGF-beta) superfamily, like growthand differentiation factor 5 (GDF-5), are shown to protect tyrosinehydroxylase or dopaminergic neurones from apoptosis. CRIB (cellularreplacement by immunoisolatory biocapsule) is a gene therapeuticalapproach for human NGF secretion, which has been shown to protectcholinergic neurones from cell death when implanted in the brain ((2000)Expert Opin Investig Drugs 9(4):747-64).

[0229] Provided herein is a method for predicting a response of asubject to an agent used to treat an A2M-mediated disease which includesa step of determining in nucleic acid obtained from the subject theidentity of nucleotide(s) at one or more polymorphisms of an A2M genethat occur at positions corresponding to 6i, 12i.1, 12i.2, 12e, 14e,14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i, and 30e, wherein the presenceor absence of a particular nucleotide(s) at the one or morepolymorphisms, individually and/or in combination, is indicative of anincreased or decreased likelihood that the treatment will be effective.Also provided are methods for predicting a response of a subject to anagent used to treat a neurodegenerative disease or disorder whichinclude a step of determining in nucleic acid obtained from the subject,the identity of nucleotide(s) at one or more polymorphisms of an A2Mgene that occur at positions corresponding to 6i, 12i.1, 12i.2, 12e,14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i, and 30e, wherein thepresence or absence of a particular nucleotide(s) at the one or morepolymorphisms, individually and/or in combination, is indicative of anincreased or decreased likelihood that the treatment will be effective.

[0230] Also provided are any of the above methods wherein theneurodegenerative disease or disorder is Alzheimer's disease. Inparticular methods, the neurodegenerative disease or disorder isAlzheimer's disease wherein the age of onset is greater than or equal toabout 50 years, or greater than or equal to about 60 years, or greaterthan or equal to about 65 years.

[0231] Also provided are any of the above methods which include a stepof determining the identity of a nucleotide(s) at a positioncorresponding to the position of at least one polymorphism of at leastone different gene, wherein the different gene is associated with aneurodegenerative disease or disorder. For example, the at least onedifferent gene can be APOE4.

[0232] As set forth above, the ability to predict whether a person willrespond to a particular therapeutic agent or drug is useful, among otherthings, for matching particular drug treatments to particular patientpopulation to thereby eliminate from a treatment protocol drugs that maybe less efficacious in particular patients.

[0233] Provided herein is a computer-assisted method of identifying aproposed treatment for a disease, such as, for example, aneurodegenerative disease. The method involves the steps of (a) storinga database of biological data for a plurality of subjects, thebiological data that is being stored include for each of the pluralityof subjects (i) treatment type, (ii) the presence or absence of aparticular nucleotide(s) at one or more polymorphisms of the A2M geneselected from the group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1,14i.2, 17i.1, 20e, 20i, 21i, 28i, and 30e, and (iii) at least onedisease progression measure for the neurodegenerative disease (e.g.,AD), or other disease, from which treatment efficacy may be determined;and then (b) querying the database to determine the dependence on theone or more polymorphisms of the effectiveness of a treatment type intreating the disease, to thereby identify a proposed treatment as aneffective treatment for a subject carrying a particular polymorphism (orcombination of polymorphisms) for the disease, such as AD. Thepolymorphisms entered into the database can also include previouslyknown polymorphisms, including, for example, polymorphisms included inTable 2.

[0234] Any suitable disease progression measure can be used. Forexample, for neurodegenerative disease, measures of motor function,cognitive function, dementia and combinations thereof can be used asmeasures of disease progression. The measures can be scored inaccordance with standard techniques for entry into the database.Measures can be taken at the initiation of the study, and then duringthe course of the study (that is, treatment of the group of patientswith the experimental and control treatments), and the database canincorporate a plurality of these measures taken over time so that thepresence, absence or rate of disease progression in particularindividuals or groups of individuals may be assessed. The database canbe queried for the effectiveness of a particular treatment in patientscarrying any of a variety of polymorphisms, or combinations ofpolymorphisms, or who lack particular polymorphisms. Computer systemsused to carry out these methods may be implemented as hardware,software, or both hardware and software. Systems that may be used toimplement these methods are known and available. See, e.g., U.S. Pat.No. 6,108,635 and Eas, M. A.: A program for the meta-analysis ofclinical trials, Computer Methods and Programs in Biomedicine, vol. 53,no. 3 (July 1997); D. Klinger and M. Jaffe, An Information TechnologyArchitecture for Pharmaceutical Research and Development, 14^(th) AnnualSymposium on Computer Applications in Medical Care, Nov. 4-7, pp.256-260 (Washington D.C., 1990); M. Rosenberg, “ClinAccess: Anintegrated client/server approach to clinical data management andregulatory approval,” Proc. Of the 21^(st) Annual SAS Users GroupInternational Conference (Cary, N.C. , Mar. 10-13, 1996). Querying ofthe database may be carried out in accordance with known techniques suchas regression analysis or other types of comparisons such as with simplenormal or t-tests, or with non-parametric techniques. Such querying maybe carried out prospectively or retrospectively on the database by anysuitable means, but is generally done by statistical analysis inaccordance with known techniques.

[0235] Rational Drug Design

[0236] Rational drug design involving polypeptides requires identifyingand defining a first peptide with which the designed drug is tointeract, and using the first target peptide to define the requirementsfor a second peptide. With such requirements defined, one can find orprepare an appropriate peptide or non-peptide that meets all orsubstantially all of the defined requirements. Thus, one goal ofrational drug design is to produce structural or functional analogs ofbiologically active polypeptides of interest or of small molecules withwhich they interact (e.g., agonists, antagonists, null compounds) inorder to fashion drugs that are, for example, more or less potent formsof the ligand. (See, e.g., Hodgson, Bio. Technology 9:19-21 (1991)). Anexample of rational drug design is shown in Erickson et al., Science249:527-533 (1990). Combinatorial chemistry is the science ofsynthesizing and testing compounds for bioactivity en masse, instead ofone by one, the aim being to discover drugs and materials more quicklyand inexpensively than was formerly possible. Rational drug design andcombinatorial chemistry have become more intimately related in recentyears due to the development of approaches in computer-aided proteinmodeling and drug discovery. (See e.g., U.S. Pat. No. 4,908,773;5,884,230; 5,873,052; 5,331,573; and 5,888,738).

[0237] The use of molecular modeling as a tool for rational drug designand combinatorial chemistry has dramatically increased due to the adventof computer graphics. Not only is it possible to view molecules oncomputer screens in three dimensions but it is also possible to examinethe interactions of macromolecules such as enzymes and receptors andrationally design derivative molecules to test. (See Boorman, Chem. Eng.News 70:18-26 (1992). A vast amount of user-friendly software andhardware is now available and virtually all pharmaceutical companieshave computer modeling groups devoted to rational drug design. MolecularSimulations Inc., for example, sells several sophisticated programs thatallow a user to start from an amino acid sequence, build a two orthree-dimensional model of the protein or polypeptide, compare it toother two and three-dimensional models, and analyze the interactions ofcompounds, drugs, and peptides with a three dimensional model in realtime. Accordingly, in some embodiments of the invention, software isused to compare regions of polymorphic and/or mutant A2M polypeptidesand molecules that interact with polymorphic and/or mutant A2Mpolypeptides (collectively referred to as “binding partners”) with othermolecules, such as peptides, peptidomimetics, and chemicals, so thattherapeutic interactions can be predicted and designed. (See Schneider,Genetic Engineering News December: page 20 (1998), Tempczyk et al.,Molecular Simulations Inc. Solutions April (1997) and Butenhof,Molecular Simulations Inc. Case Notes (August 1998) for a discussion ofmolecular modeling).

[0238] For example, the protein sequence of a polymorphic and/or mutantA2M polypeptide or binding partner, or domains of these molecules (ornucleic acid sequence encoding these polypeptides or both), can beentered onto a computer readable medium for recording and manipulation.It will be appreciated by those skilled in the art that a computerreadable medium having these sequences can interface with software thatconverts or manipulates the sequences to obtain structural andfunctional information, such as protein models. That is, thefunctionality of a software program that converts or manipulates thesesequences includes the ability to compare these sequences to othersequences or structures of molecules that are present on publicly andcommercially available databases so as to conduct rational drug design.

[0239] The polymorphic and/or mutant A2M polypeptide or binding partnerpolypeptide or nucleic acid sequence or both can be stored, recorded,and manipulated on any medium that can be read and accessed by acomputer. As used herein, the words “recorded” and “stored” refer to aprocess for storing information on computer readable medium. A skilledartisan can readily adopt any of the presently known methods forrecording information on a computer readable medium to generatemanufactures comprising the nucleotide or polypeptide sequenceinformation of this embodiment. A variety of data storage structures areavailable to a skilled artisan for creating a computer readable mediumhaving recorded thereon a nucleotide or polypeptide sequence. The choiceof the data storage structure will generally be based on the componentchosen to access the stored information. Computer readable media includemagnetically readable media, optically readable media, or electronicallyreadable media. For example, the computer readable media can be a harddisc, a floppy disc, a magnetic tape, zip disk, CD-ROM, DVD-ROM, RAM, orROM as well as other types of other media known to those skilled in theart. The computer readable media on which the sequence information isstored can be in a personal computer, a network, a server or othercomputer systems known to those skilled in the art.

[0240] Embodiments of the invention utilize computer-based systems thatcontain the sequence information described herein and convert thisinformation into other types of usable information (e.g., protein modelsfor rational drug design). The term “a computer-based system” refers tothe hardware, software, and any database used to analyze an polymorphicand/or mutant A2M or a binding partner (nucleic acid or polypeptidesequence or both), or fragments of these biomolecules so as to constructmodels or to conduct rational drug design. The computer-based systempreferably includes the storage media described above, and a processorfor accessing and manipulating the sequence data. The hardware of thecomputer-based systems of this embodiment comprise a central processingunit (CPU) and a database. A skilled artisan can readily appreciate thatany one of the currently available computer-based systems are suitable.

[0241] In one particular embodiment, the computer system includes aprocessor connected to a bus that is connected to a main memory(preferably implemented as RAM) and a variety of secondary storagedevices, such as a hard drive and removable medium storage device. Theremovable medium storage device can represent, for example, a floppydisk drive, a DVD drive, an optical disk drive, a compact disk drive, amagnetic tape drive, etc. A removable storage medium, such as a floppydisk, a compact disk, a magnetic tape, etc. containing control logicand/or data recorded therein can be inserted into the removable storagedevice. The computer system includes appropriate software for readingthe control logic and/or the data from the removable medium storagedevice once inserted in the removable medium storage device. Thepolymorphic and/or mutant A2M or binding partner (nucleic acid orpolypeptide sequence or both) can be stored in a well known manner inthe main memory, any of the secondary storage devices, and/or aremovable storage medium. Software for accessing and processing thesesequences (such as search tools, compare tools, and modeling tools etc.)reside in main memory during execution.

[0242] As used herein, “a database” refers to memory that can store apolymorphic and/or mutant A2M or binding partner nucleotide orpolypeptide sequence information, protein model information, informationon other peptides, chemicals, peptidomimetics, and other agents thatinteract with polymorphic and/or mutant A2M polypeptides, and values orresults from functional assays. Additionally, a “database” refers to amemory access component that can access manufactures having recordedthereon polymorphic and/or mutant A2M or binding partner nucleotide orpolypeptide sequence information, protein model information, informationon other peptides, chemicals, peptidomimetics, and other agents thatinteract with polymorphic and/or mutant A2M polypeptides, and values orresults from functional assays. In other embodiments, a database storesa “polymorphic and/or mutant A2M polypeptide functional profile”comprising the values and results (e.g., ability to associate with areceptyor, amyloid, β, a protease, zinc, or the ability to form atetramer) from one or more “A2M functional assays”, as described hereinor known in the art, and relationships between these values or results.The sequence data and values or results from these functional assays canbe stored and manipulated in a variety of data processor programs in avariety of formats. For example, the sequence data can be stored as textin a word processing file, such as Microsoft WORD or WORDPERFECT, anASCII file, a html file, or a pdf file in a variety of database programsfamiliar to those of skill in the art, such as DB2, SYBASE, or ORACLE.

[0243] A “search program” refers to one or more programs that areimplemented on the computer-based system to compare a polymorphic and/ormutant A2M or binding partner (nucleotide or polypeptide sequence) withother nucleotide or polypeptide sequences and agents including but notlimited to peptides, peptidomimetics, and chemicals stored within adatabase. A search program also refers to one or more programs thatcompare one or more protein models to several protein models that existin a database and one or more protein models to several peptides,peptidomimetics, and chemicals that exist in a database. A searchprogram is used, for example, to compare one polymorphic and/or mutantA2M functional profile to one or more polymorphic and/or mutant A2Mfunctional profiles that are present in a database so as to determine anappropriate treatment protocol, for example. Still further, a searchprogram can be used to compare values or results from A2M functionalassays and agents that modulate A2M-mediated activities.

[0244] A “retrieval program” refers to one or more programs that can beimplemented on the computer-based system to identify peptides,peptidomimetics, and chemicals that interact with a polymorphic and/ormutant A2M polypeptide sequence, or a polymorphic and/or mutant A2Mpolypeptide model stored in a database. Further, a retrieval program isused to identify a specific agent that modulates A2M-mediated activitiesto a desired set of values, results, or profile. That is, a retrievalprogram can also be used to obtain “a binding partner profile” that iscomposed of a chemical structure, nucleic acid sequence, or polypeptidesequence or model of an agent that interacts with a polymorphic and/ormutant A2M polypeptide and, thereby modulates (inhibits or enhances) anA2M activity, such as binding to a receptor, amyloid β, a protease.zinc, or tetramer formation. Further, a binding partner profile can haveone or more symbols that represent these molecules and/or models, anidentifier that represents one or more agents including, but not limitedto peptides and peptidomimetics (referred to collectively as “peptideagents”) and chemicals, and a value or result from a functional assay.

[0245] As a starting point to rational drug design, a two or threedimensional model of a polypeptide of interest is created (e.g.,polymorphic and/or mutant A2M polypeptide, or a binding partner, such asthe LRP receptor, amyloid β, a protease, or an antibody). In the past,the three-dimensional structure of proteins has been determined in anumber of ways. Perhaps the best known way of determining proteinstructure involves the use of x-ray crystallography. A general review ofthis technique can be found in Van Holde, K. E. Physical Biochemistry,Prentice-Hall, N.J. pp. 221-239 (1971). Using this technique, it ispossible to elucidate three-dimensional structure with good precision.Additionally, protein structure can be determined through the use oftechniques of neutron diffraction, or by nuclear magnetic resonance(NMR). (See, e.g., Moore, W. J., Physical Chemistry, 4^(th) Edition,Prentice-Hall, N.J. (1972)).

[0246] Alternatively, protein models of a polypeptide of interest can beconstructed using computer-based protein modeling techniques. By oneapproach, the protein folding problem is solved by finding targetsequences that are most compatible with profiles representing thestructural environments of the residues in known three-dimensionalprotein structures. (See, e.g., U.S. Pat. No. 5,436,850). In anothertechnique, the known three-dimensional structures of proteins in a givenfamily are superimposed to define the structurally conserved regions inthat family. This protein modeling technique also uses the knownthree-dimensional structure of a homologous protein to approximate thestructure of a polypeptide of interest. (See e.g., U.S. Pat. Nos.5,557,535; 5,884,230; and 5,873,052). Conventional homology modelingtechniques have been used routinely to build models of proteases andantibodies. (Sowdhamini et al., Protein Engineering 10:207, 215 (1997)).Comparative approaches can also be used to develop three-dimensionalprotein models when the protein of interest has poor sequence identityto template proteins. In some cases, proteins fold into similarthree-dimensional structures despite having very weak sequenceidentities. For example, the three-dimensional structures of a number ofhelical cytokines fold in similar three-dimensional topology in spite ofweak sequence homology.

[0247] The recent development of threading methods and “fuzzy”approaches now enables the identification of likely folding patterns andfunctional protein domains in a number of situations where thestructural relatedness between target and template(s) is not detectableat the sequence level. By one method, fold recognition is performedusing Multiple Sequence Threading (MST) and structural equivalences arededuced from the threading output using the distance geometry programDRAGON that constructs a low resolution model. A full-atomrepresentation is then constructed using a molecular modeling packagesuch as QUANTA.

[0248] According to this 3-step approach, candidate templates are firstidentified by using the novel fold recognition algorithm MST, which iscapable of performing simultaneous threading of multiple alignedsequences onto one or more 3-D structures. In a second step, thestructural equivalences obtained from the MST output are converted intointerresidue distance restraints and fed into the distance geometryprogram DRAGON, together with auxiliary information obtained fromsecondary structure predictions. The program combines the restraints inan unbiased manner and rapidly generates a large number of lowresolution model confirmations. In a third step, these low resolutionmodel confirmations are converted into full-atom models and organized toenergy minimization using the molecular modeling package QUANTA. (Seee.g., Aszódi et al., Proteins:Structure, Function, and Genetics,Supplement 1:38-42 (1997)).

[0249] In a preferred approach, the commercially available “Insight II98” program (Molecular Simulations Inc.) and accompanying modules areused to create a two and/or three dimensional model of a polypeptide ofinterest from an amino acid sequence. Insight II is a three-dimensionalgraphics program that can interface with several modules that performnumerous structural analysis and enable real-time rational drug designand combinatorial chemistry. Modules such as Builder, Biopolymer,Consensus, and Converter, for example, allow one to rapidly create a twodimensional or three dimensional model of a polypeptide, carbohydrate,nucleic acid, chemical or combinations of the foregoing from theirsequence or structure. The modeling tools associated with Insight IIsupport many different data file formats including Brookhaven andCambridge databases; AMPAC/MOPAC and QCPE programs; Molecular DesignLimited Molfile and SD files, Sybel Mol2 files, VRML, and Pict files.

[0250] Additionally, the techniques described above can be supplementedwith techniques in molecular biology to design models of the protein ofinterest. For example, a polypeptide of interest can be analyzed by analanine scan (Wells, Methods in Enzymol. 202:390-411 (1991)) or othertypes of site-directed mutagenesis analysis. In alanine scan, each aminoacid residue of the polypeptide of interest is sequentially replaced byalanine in a step-wise fashion (i.e., only one alanine point mutation isincorporated per molecule starting at position #1 and proceeding throughthe entire molecule), and the effect of the mutation on the peptide'sactivity in a functional assay is determined. Each of the amino acidresidues of the peptide is analyzed in this manner and the regionsimportant for A2M activities, are identified. These functionallyimportant regions can be recorded on a computer readable medium, storedin a database in a computer system, and a search program can be employedto generate a protein model of the functionally important regions.

[0251] Once a model of the polypeptide of interest is created, acandidate binding partner can be identified and manufactured as follows.First, a molecular model of one or more molecules that are known tointeract with A2M or portions thereof are created using one of thetechniques discussed above or as known in the art. Next, chemicallibraries and databases are searched for molecules similar in structureto the known molecule. That is, a search can be made of a threedimensional data base for non-peptide (organic) structures (e.g.,non-peptide analogs, and/or dipeptide analogs) having three dimensionalsimilarity to the known structure of the target compound. See, e.g., theCambridge Crystal Structure Data Base, Crystallographic Data Center,Lensfield Road, Cambridge, CB2 1EW, England; and Allen, F. H., et al.,Acta Crystallogr., B35: 2331-2339 (1979). The identified candidatebinding partners that interact with A2M can then be analyzed in afunctional assay (e.g., binding assays with amyloid β, the LRP receptor,zinc, protease, or tetramer formation) and new molecules can be modeledafter the candidate binding partners that produce a desirable response.Preferably, these interactions are studied with both wild-type A2M andpolymorphic and/or mutant A2M polypeptides. By cycling in this fashion,libraries of molecules that interact with A2M, preferably polymorphicand/or mutant A2M polypeptides, and produce a desirable or optimalresponse in a functional assay can be selected.

[0252] It is noted that search algorithms for three dimensional database comparisons are available in the literature. See, e.g., Cooper, etal., J. Comput. -Aided Mol. Design, 3: 253-259 (1989) and referencescited therein; Brent, et al., J. Comput.-Aided Mol. Design, 2: 311-310(1988) and references cited therein. Commercial software for suchsearches is also available from vendors such as Day Light InformationSystems, Inc., Irvine, Calif. 92714, and Molecular Design Limited, 2132Faralton Drive, San Leandro, Calif. 94577. The searching is done in asystematic fashion by simulating or synthesizing analogs having asubstitute moiety at every residue level. Preferably, care is taken thatreplacement of portions of the backbone does not disturb the tertiarystructure and that the side chain substitutions are compatible to retainthe receptor substrate interactions.

[0253] By another approach, protein models of binding partners thatinteract with A2M, preferably polymorphic and/or mutant A2Mpolypeptides, can be made by the methods described above and thesemodels can be used to predict the interaction of new molecules. Once amodel of a binding partner is identified, the active sites or regions ofinteraction can be identified. Such active sites might typically beligand binding sites. The active site can be identified using methodsknown in the art including, for example, from the amino acid sequencesof peptides, from the nucleotide sequences of nucleic acids, or fromstudy of complexes of the wild-type and/or polymorphic and/or mutant A2Mpolypeptides with a ligand. In the latter case, chemical or X-raycrystallographic methods can be used to find the active site by findingwhere on the wild-type and/or polymorphic and/or mutant A2M polypeptidesthe complexed ligand is found. Next, the three dimensional geometricstructure of the active site is determined. This can be done by knownmethods, including X-ray crystallography, which can determine a completemolecular structure. On the other hand, solid or liquid phase NMR can beused to determine certain intra-molecular distances. Any otherexperimental method of structure determination can be used to obtainpartial or complete geometric structures. The geometric structures canbe measured with a complexed ligand, natural or artificial, which mayincrease the accuracy of the active site structure determined.

[0254] If an incomplete or insufficiently accurate structure isdetermined, the methods of computer based numerical modeling can be usedto complete the structure or improve its accuracy. Any recognizedmodeling method can be used, including parameterized models specific toparticular biopolymers such as proteins or nucleic acids, moleculardynamics models based on computing molecular motions, statisticalmechanics models based on thermal ensembles, or combined models. Formost types of models, standard molecular force fields, representing theforces between constituent atoms and groups, are necessary, and can beselected from force fields known in physical chemistry. The incompleteor less accurate experimental structures can serve as constraints on thecomplete and more accurate structures computed by these modelingmethods.

[0255] Finally, having determined the structure of the active site ofthe known binding partner, either experimentally, by modeling, or by acombination, candidate binding partners can be identified by searchingdatabases containing compounds along with information on their molecularstructure. Such a search seeks compounds having structures that matchthe determined active site structure and that interact with the groupsdefining the active site. Such a search can be manual, but is preferablycomputer assisted. One program that allows for such analysis is InsightII having the Ludi module. Further, the Ludi/ACD module allows a useraccess to over 65,000 commercially available drug candidates (MDL'sAvailable Chemicals Directory) and provides the ability to screen thesecompounds for interactions with the protein of interest.

[0256] Alternatively, these methods can be used to identify improvedbinding partners from an already known binding partner. The compositionof the known binding partner can be modified and the structural effectsof modification can be determined using the experimental and computermodeling methods described above applied to the new composition. Thealtered structure is then compared to the active site structure of thecompound to determine if an improved fit or interaction results. In thismanner systematic variations in composition, such as by varying sidegroups, can be quickly evaluated to obtain modified modulating compoundsor ligands of improved specificity or activity.

[0257] A number of articles review computer modeling of drugsinteractive with specific-proteins, such as Rotivinen, et al., 1988,Acta Pharmaceutical Fennica 97:159-166; Ripka, New Scientist 54-57 (Jun.16, 1988); McKinaly and Rossmann, 1989, Annu. Rev. Pharmacol. Toxiciol.29:111-122; Perry and Davies, OSAR: Quantitative Structure-ActivityRelationships in Drug Design pp. 189-193 (Alan R. Liss, Inc. 1989);Lewis and Dean, 1989 Proc. R. Soc. Lond. 236:125-140 and 141-162; and,with respect to a model receptor for nucleic acid components, Askew, etal., 1989, J. Am. Chem. Soc. 111:1082-1090. Other computer programs thatscreen and graphically depict chemicals are available from companiessuch as BioDesign, Inc. (Pasadena, Calif.), Allelix, Inc. (Mississauga,Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario). Althoughthese are primarily designed for application to drugs specific toparticular proteins, they can be adapted to design of drugs specific forthe modulation of A2M activities.

[0258] Many more computer programs and databases can be used withembodiments of the invention to identify new binding partners thatmodulate A2M function. The following list is intended not to limit theinvention but to provide guidance to programs and databases that areuseful with the approaches discussed above. The programs and databasesthat can be used include, but are not limited to: MacPattern (EMBL),DiscoveryBase (Molecular Applications Group), GeneMine (MolecularApplications Group), Look (Molecular Applications Group), MacLook(Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN andBLASTX (Altschul et al, J. Mol. Biol. 215: 403 (1990), hereinincorporated by reference), FASTA (Pearson and Lipman, Proc. Natl. Acad.Sci. USA, 85: 2444 (1988), herein incorporated by reference), Catalyst(Molecular Simulations Inc.), Catalyst/SHAPE (Molecular SimulationsInc.), Cerius².DBAccess (Molecular Simulations Inc.), HypoGen (MolecularSimulations Inc.), Insight II, (Molecular Simulations Inc.), Discover(Molecular Simulations Inc.), CHARMm (Molecular Simulations Inc.), Felix(Molecular Simulations Inc.), DelPhi, (Molecular Simulations Inc.),QuanteMM, (Molecular Simulations Inc.), Homology (Molecular SimulationsInc.), Modeler (Molecular Simulations Inc.), Modeller 4 (SalI andBlundell J. Mol. Biol. 234:217-241 (1997)), ISIS (Molecular SimulationsInc.), Quanta/Protein Design (Molecular Simulations Inc.), WebLab(Molecular Simulations Inc.), WebLab Diversity Explorer (MolecularSimulations Inc.), Gene Explorer (Molecular Simulations Inc.), SeqFold(Molecular Simulations Inc.), Biopendium (Inpharmatica), SBdBase(Structural Bioinformatics), the EMBL/Swissprotein database, the MDLAvailable Chemicals Directory database, the MDL Drug Data Report database, the Comprehensive Medicinal Chemistry database, Derwents's WorldDrug Index database, and the BioByteMasterFile database. Many otherprograms and data bases would be apparent to one of skill in the artgiven the present disclosure.

[0259] Once candidate binding partners have been identified, desirably,they are analyzed in a functional assay. Further cycles of modeling andfunctional assays can be employed to more narrowly define the parametersneeded in a binding partner. Each binding partner and its response in afunctional assay can be recorded on a computer readable media and adatabase or library of binding partners and respective responses in afunctional assay can be generated. These databases or libraries can beused by researchers to identify important differences between active andinactive molecules so that compound libraries are enriched for bindingpartners that have favorable characteristics. The section belowdescribes several A2M functional assays that can be used to characterizeA2M interactions with candidate binding partners.

[0260] A2M Characterization Assays

[0261] The term “A2M characterization assay” or “A2M functional assay”or “functional assay” the results of which can be recorded as a value ina “A2M functional profile”, include assays that directly or indirectlyevaluate the presence of an A2M nucleic acid or protein in a cell andthe ability of a particular type of A2M polypeptide, in particularpolymorphic and/or mutant A2M polypeptides, to associate with areceptor, a protease, amyloid β, zinc, or to form a tetramer.

[0262] Some functional assays involve binding assays that utilizemultimeric agents. One form of multimeric agent concerns a manufacturecomprising an polymorphic and/or mutant A2M polypeptide disposed on asupport. These multimeric agents provide the polypeptide in such a formor in such a way that a sufficient affinity for its ligand is achieved.A multimeric agent having an polymorphic and/or mutant A2M polypeptideis obtained by joining the desired polypeptide to a macromolecularsupport. A “support” can be a termed a carrier, a protein, a resin, acell membrane, or any macromolecular structure used to join orimmobilize such molecules. Solid supports include, but are not limitedto, the walls of wells of a reaction tray, test tubes, polystyrenebeads, magnetic beads, nitrocellulose strips, membranes, microparticlessuch as latex particles, animal cells, Duracyteo®, artificial cells, andothers. A polymorphic and/or mutant A2M polypeptide can also be joinedto inorganic carriers, such as silicon oxide material (e.g., silica gel,zeolite, diatomaceous earth or aminated glass) by, for example, acovalent linkage through a hydroxy, carboxy or amino group and areactive group on the carrier.

[0263] In several multimeric agents, the macromolecular support has ahydrophobic surface that interacts with a portion of the polymorphicand/or mutant A2M polypeptides by a hydrophobic non-covalentinteraction. In some cases, the hydrophobic surface of the support is apolymer such as plastic or any other polymer in which hydrophobic groupshave been linked such as polystyrene, polyethylene or polyvinyl.Additionally, polymorphic and/or mutant A2M polypeptides can becovalently bound to carriers including proteins and oligo/polysaccarides(e.g. cellulose, starch, glycogen, chitosane or aminated sepharose). Inthese later multimeric agents, a reactive group on the molecule, such asa hydroxy or an amino group, is used to join to a reactive group on thecarrier so as to create the covalent bond. Additional multimeric agentscomprise a support that has other reactive groups that are chemicallyactivated so as to attach the polymorphic and/or mutant A2Mpolypeptides. For example, cyanogen bromide activated matrices, epoxyactivated matrices, thio and thiopropyl gels, nitrophenyl chloroformateand N-hydroxy succinimide chlorformate linkages, or oxirane acrylicsupports are used. (Sigma).

[0264] Furthermore, in some embodiments, a liposome or lipid bilayer(natural or synthetic) is contemplated as a support and polymorphicand/or mutant A2M polypeptides, or binding partners are attached to themembrane surface or are incorporated into the membrane by techniques inliposome engineering. Carriers for use in the body, (i.e. forprophylactic or therapeutic applications) are desirably physiological,non-toxic and preferably, non-immunoresponsive. Suitable carriers foruse in the body include poly-L-lysine, poly-D, L-alanine, liposomes, andChromosorb® (Johns-Manville Products, Denver Co.). Ligand conjugatedChromosorb® (Synsorb-Pk) has been tested in humans for the prevention ofhemolytic-uremic syndrome and was reported as not presenting adversereactions. (Armstrong et al. J. Infectious Diseases 171:1042-1045(1995)).

[0265] The insertion of linkers, such as linkers (e.g., “λ linkers”engineered to resemble the flexible regions of λ phage) of anappropriate length between the polymorphic and/or mutant A2Mpolypeptides and the support are also contemplated so as to encouragegreater flexibility and thereby overcome any steric hindrance that canbe presented by the support. The determination of an appropriate lengthof linker that allows for an optimal cellular response or lack thereof,can be determined by screening the polymorphic and/or mutant A2Mpolypeptides with varying linkers in the assays detailed in the presentdisclosure.

[0266] A composite support comprising more than one type of polymorphicand/or mutant A2M polypeptides is also envisioned. A “composite support”can be a carrier, a resin, or any macromolecular structure used toattach or immobilize two or more different binding partners orpolymorphic and/or mutant A2M polypeptides. In some embodiments, aliposome or lipid bilayer (natural or synthetic) is contemplated for usein constructing a composite support and polymorphic and/or mutant A2Mpolypeptides or binding partners are attached to the membrane surface orare incorporated into the membrane using techniques in liposomeengineering.

[0267] As above, the insertion of linkers, such as λ linkers, of anappropriate length between the polymorphic and/or mutant A2Mpolypeptides or binding partner and the support is also contemplated soas to encourage greater flexibility in the molecule and thereby overcomeany steric hindrance that can occur. The determination of an appropriatelength of linker that allows for an optimal cellular response or lackthereof, can be determined by screening the polymorphic and/or mutantA2M polypeptides or binding partners with varying linkers in the assaysdetailed in the present disclosure.

[0268] In other embodiments of the invention, the multimeric andcomposite supports discussed above can have attached multimerizedpolymorphic and/or mutant A2M polypeptides, or binding partners so as tocreate a “multimerized-multimeric support” and a “multimerized-compositesupport”, respectively. A multimerized ligand can, for example, beobtained by coupling two or more binding partners in tandem usingconventional techniques in molecular biology. The multimerized form ofthe polymorphic and/or mutant A2M polypeptides, or binding partner canbe advantageous for many applications because of the ability to obtainan agent with a higher affinity for A2M, for example. The incorporationof linkers or spacers, such as flexible λ linkers, between theindividual domains that make-up the multimerized agent can also beadvantageous for some embodiments. The insertion of λ linkers of anappropriate length between protein binding domains, for example, canencourage greater flexibility in the molecule and can overcome sterichindrance. Similarly, the insertion of linkers between the multimerizedbinding partner or polymorphic and/or mutant A2M polypeptides and thesupport can encourage greater flexibility and limit steric hindrancepresented by the support. The determination of an appropriate length oflinker can be determined by screening the polymorphic and/or mutant A2Mpolypeptides and binding partners with varying linkers in the assaysdetailed in this disclosure.

[0269] Thus, several approaches to identify agents that interact with apolymorphic and/or mutant A2M polypeptide, employ a polymorphic and/ormutant A2M polypeptide joined to a support. Once the support-boundpolypeptide is obtained, for example, candidate binding partners arecontacted to the support-bound polypeptide and an association isdetermined directly (e.g., by using labeled binding partner) orindirectly (e.g., by using a labeled antibody directed to the bindingpartner). Candidate binding partners are identified as binding partnersby virtue of the association with the support-bound polypeptide. Theproperties of the binding partners are analyzed and derivatives are madeusing rational drug design and combinatorial chemistry. Candidatebinding partners can be obtained from random chemical or peptidelibraries but, preferably, are rationally selected. For example,monoclonal antibodies that bind to polymorphic and/or mutant A2Mpolypeptides can be created and the nucleic acids encoding the VH and VLdomains of the antibodies can be sequenced. These sequences can then beused to synthesize peptides that bind to the polymorphic and/or mutantA2M polypeptides. Further, peptidomimetics corresponding to thesesequences can be created. These molecules can then be used as candidatebinding partners.

[0270] Additionally, a cell based approach can be used characterizepolymorphic and/or mutant A2M polypeptides or to rapidly identifybinding partners that interact with said polypeptides and, thereby,modulate A2M activities. Preferably, molecules identified in thesupport-bound A2M assay described above are used in the cell basedapproach, however, randomly generated compounds can also be used.

[0271] Many A2M characterization assays take advantage of techniques inmolecular biology that are employed to discover protein:proteininteractions. One method that detects protein-protein interactions invivo, the two-hybrid system, is described in detail for illustrationonly and not by way of limitation. Other similar assays that can be canbe adapted to identify binding partners include:

[0272] (1) the two-hybrid systems (Field & Song, Nature 340:245-246(1989); Chien et al., Proc. Natl. Acad. Sci. USA 88:9578-9582 (1991);and Young K H, Biol. Reprod. 58:302-311 (1998), all references hereinexpressly incorporated by reference);

[0273] (2) reverse two-hybrid system (Leanna & Hannink, Nucl. Acid Res.24:3341-3347 (1996), herein incorporated by reference);

[0274] (3) repressed transactivator system (Sadowski et al., U.S. Pat.No. 5,885,779), herein incorporated by reference);

[0275] (4) phage display (Lowman H B, Annu. Rev. Biophys. Biomol.Struct. 26:401-424 (1997), herein incorporated by reference); and

[0276] (5) GST/HIS pull down assays, mutant operators (Granger et al.,WO 98/01879) and the like (See also Mathis G., Clin. Chem. 41:139-147(1995); Lam K. S. Anticancer Drug Res., 12:145-167 (1997); and Phizickyet al., Microbiol. Rev. 59:94-123 (1995), all references hereinexpressly incorporated by reference).

[0277] An adaptation of the system described by Chien et al., 1991,Proc. Natl. Acad. Sci. USA, 88:9578-9582, herein incorporated byreference), which is commercially available from Clontech (Palo Alto,Calif.) is as follows. Plasmids are constructed that encode two hybridproteins: one plasmid consists of nucleotides encoding the DNA-bindingdomain of a transcription activator protein fused to a nucleotidesequence encoding a polymorphic and/or mutant A2M polypeptide, and theother plasmid consists of nucleotides encoding the transcriptionactivator protein's activation domain fused to a cDNA encoding anunknown protein that has been recombined into this plasmid as part of acDNA library. The DNA-binding domain fusion plasmid and the cDNA libraryare transformed into a strain of the yeast Saccharomyces cerevisiae thatcontains a reporter gene (e.g., HBS or lacZ) whose regulatory regioncontains the transcription activator's binding site. Either hybridprotein alone cannot activate transcription of the reporter gene: theDNA-binding domain hybrid cannot because it does not provide activationfunction and the activation domain hybrid cannot because it cannotlocalize to the activator's binding sites. Interaction of the two hybridproteins reconstitutes the functional activator protein and results inexpression of the reporter gene, which is detected by an assay for thereporter gene product.

[0278] The two-hybrid system or related methodology can be used toscreen activation domain libraries for proteins that interact with the“bait” gene product. By way of example, and not by way of limitation,polymorphic and/or mutant A2M polypeptides can be used as the bait geneproduct. Total genomic or cDNA sequences are fused to the DNA encodingan activation domain. This library and a plasmid encoding a hybrid of abait gene encoding the polymorphic and/or mutant A2M polypeptide fusedto the DNA-binding domain are cotransformed into a yeast reporterstrain, and the resulting transformants are screened for those thatexpress the reporter gene. For example, and not by way of limitation, abait gene sequence encoding a polymorphic and/or mutant A2M polypeptidecan be cloned into a vector such that it is translationally fused to theDNA encoding the DNA-binding domain of the GAL4 protein. These coloniesare purified and the library plasmids responsible for reporter geneexpression are isolated. DNA sequencing is then used to identify theproteins encoded by the library plasmids.

[0279] A cDNA library of the cell line from which proteins that interactwith bait polymorphic and/or mutant A2M polypeptides are to be detectedcan be made using methods routinely practiced in the art. According tothe particular system described herein, for example, the cDNA fragmentscan be inserted into a vector such that they are translationally fusedto the transcriptional activation domain of GAL4. This library can beco-transformed along with the bait polymorphic and/or mutant A2Mgene-GAL4 fusion plasmid into a yeast strain which contains a lacZ genedriven by a promoter which contains GAL4 activation sequence. A cDNAencoded protein, fused to GAL4 transcriptional activation domain, thatinteracts with bait A2M gene product will reconstitute an active GAL4protein and thereby drive expression of the lacZ gene. Colonies thatexpress lacZ can be detected and the cDNA can then be purified fromthese strains, and used to produce and isolate the binding partner bytechniques routinely practiced in the art. The examples below describepreferred A2M characterization assays.

[0280] Pharmaceutical Preparations and Methods of Administration

[0281] The polymorphic and/or mutant A2M nucleic acids and polypeptidesand their binding partners are suitable for incorporation intopharmaceuticals that treat or prevent neuropathies, such as AD. Thesepharmacologically active compounds can be processed in accordance withconventional methods of galenic pharmacy to produce medicinal agents foradministration to organisms, e.g., plants, insects, mold, yeast,animals, and mammals including humans. The active ingredients can beincorporated into a pharmaceutical product with and withoutmodification. Further, the manufacture of pharmaceuticals or therapeuticagents that deliver the pharmacologically active compounds of thisinvention by several routes are aspects of the invention. For example,and not by way of limitation, DNA, RNA, and viral vectors havingsequence encoding the polymorphic and/or mutant A2M polypeptides,binding partners, or fragments thereof are used with embodiments.Nucleic acids encoding polymorphic and/or mutant A2M polypeptides orbinding partners can be administered alone or in combination with otheractive ingredients.

[0282] The compounds of this invention can be employed in admixture withconventional excipients, i.e., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, enteral (e.g.,oral) or topical application that do not deleteriously react with thepharmacologically active ingredients of this invention. Suitablepharmaceutically acceptable carriers include, but are not limited to,water, salt solutions, alcohols, gum arabic, vegetable oils, benzylalcohols, polyetylene glycols, gelatine, carbohydrates such as lactose,amylose or starch, magnesium stearate, talc, silicic acid, viscousparaffin, perfume oil, fatty acid monoglycerides and diglycerides,pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinylpyrrolidone, etc. Many more suitable vehicles are described inRemmington's Pharmaceutical Sciences, 15th Edition, Easton:MackPublishing Company, pages 1405-1412 and 1461-1487(1975) and The NationalFormulary XIV, 14th Edition, Washington, American PharmaceuticalAssociation (1975), herein incorporated by reference. The pharmaceuticalpreparations can be sterilized and if desired mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,flavoring and/or aromatic substances and the like that do notdeleteriously react with the active compounds.

[0283] The effective dose and method of administration of a particularpharmaceutical formulation having polymorphic and/or mutant A2Mpolypeptides or nucleic acids or binding partners, or fragments thereofcan vary based on the individual needs of the patient and the treatmentor preventative measure sought. Therapeutic efficacy and toxicity ofsuch compounds can be determined by standard pharmaceutical proceduresin cell cultures or experimental animals, e.g., ED50 (the dosetherapeutically effective in 50% of the population). The data obtainedfrom these assays is then used in formulating a range of dosage for usewith other organisms, including humans. The dosage of such compoundslies preferably within a range of circulating concentrations thatinclude the ED50 with no toxicity. The dosage varies within this rangedepending upon type of polymorphic and/or mutant A2M polypeptide ornucleic acid or binding partner, or fragment thereof, the dosage formemployed, sensitivity of the organism, and the route of administration.

[0284] Normal dosage amounts of various polymorphic and/or mutant A2Mpolypeptide or nucleic acid or binding partner, or fragment thereof canvary from any number between approximately 1 to 100,000 micrograms, upto a total dose of about 10 grams, depending upon the route ofadministration. Desirable dosages include, for example, 250 μg, 500 μg,1 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, ,300 mg, 350 mg, 400 mg,450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg,900 mg, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9g, 2 g, 3 g, 4 g, 5, 6 g, 7 g, 8 g, 9 g, and 10 g.

[0285] In some embodiments, the dose of polymorphic and/or mutant A2Mpolypeptide or nucleic acid or binding partner, or fragment thereofpreferably produces a tissue or blood concentration or both fromapproximately any number between 0.1 μM to 500 mM. Desirable dosesproduce a tissue or blood concentration or both of about any numberbetween 1 to 800 μM. Preferable doses produce a tissue or bloodconcentration of greater than about any number between 10 μM to about500 μM. Preferable doses are, for example, the amount of activeingredient required to achieve a tissue or blood concentration or bothof 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM,60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, 110 μM,120 μM, 130 μM, 140 μM, 145 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM,200 μM, 220 μM, 240 μM, 250 μM, 260 μM, 280 μM, 300 μM, 320 μM, 340 μM,360 μM, 380 μM, 400 μM, 420 μM, 440 μM, 460 μM, 480 μM, and 500 μM.Although doses that produce a tissue concentration of greater than 800μM are not preferred, they can be used with some embodiments of theinvention. A constant infusion of the polymorphic and/or mutant A2Mpolypeptide or nucleic acid or binding partner, or fragment thereof canalso be provided so as to maintain a stable concentration in the tissuesas measured by blood levels.

[0286] The exact dosage is chosen by the individual physician in view ofthe patient to be treated. Dosage and administration are adjusted toprovide sufficient levels of the active moiety or to maintain thedesired effect. Additional factors that can be taken into accountinclude the severity of the disease, age of the organism, and weight orsize of the organism; diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Short acting pharmaceutical compositions are administered dailywhereas long acting pharmaceutical compositions are administered every2, 3 to 4 days, every week, or once every two weeks. Depending onhalf-life and clearance rate of the particular formulation, thepharmaceutical compositions of the invention are administered once,twice, three, four, five, six, seven, eight, nine, ten or more times perday.

[0287] Routes of administration of the pharmaceuticals of the inventioninclude, but are not limited to, topical, transdermal, parenteral,gastrointestinal, transbronchial, and transalveolar. Transdermaladministration is accomplished by application of a cream, rinse, gel,etc. capable of allowing the pharmacologically active compounds topenetrate the skin. Parenteral routes of administration include, but arenot limited to, electrical or direct injection such as direct injectioninto a central venous line, intravenous, intramuscular, intraperitoneal,intradermal, or subcutaneous injection. Gastrointestinal routes ofadministration include, but are not limited to, ingestion and rectal.Transbronchial and transalveolar routes of administration include, butare not limited to, inhalation, either via the mouth or intranasally.

[0288] Compositions having the pharmacologically active compounds ofthis invention that are suitable for transdermal or topicaladministration include, but are not limited to, pharmaceuticallyacceptable suspensions, oils, creams, and ointments applied directly tothe skin or incorporated into a protective carrier Such as a transdermaldevice (“transdermal patch”). Examples of suitable creams, ointments,etc. can be found, for instance, in the Physician's Desk Reference.Examples of suitable transdermal devices are described, for instance, inU.S. Pat. No. 4,818,540 issued Apr. 4, 1989 to Chinen, et al., hereinincorporated by reference.

[0289] Compositions having the pharmacologically active compounds ofthis invention that are suitable for parenteral administration include,but are not limited to, pharmaceutically acceptable sterile isotonicsolutions. Such solutions include, but are not limited to, saline andphosphate buffered saline for injection into a central venous line,intravenous, intramuscular, intraperitoneal, intradermal, orsubcutaneous injection.

[0290] Compositions having the pharmacologically active compounds ofthis invention that are suitable for transbronchial and transalveolaradministration include, but not limited to, various types of aerosolsfor inhalation. Devices suitable for transbronchial and transalveolaradministration of these are also embodiments. Such devices include, butare not limited to, atomizers and vaporizers. Many forms of currentlyavailable atomizers and vaporizers can be readily adapted to delivercompositions having the pharmacologically active compounds of theinvention.

[0291] Compositions having the pharmacologically active compounds ofthis invention that are suitable for gastrointestinal administrationinclude, but not limited to, pharmaceutically acceptable powders, pillsor liquids for ingestion and suppositories for rectal administration.Due to the ease of use, gastrointestinal administration, particularlyoral, is a preferred embodiment. Once the pharmaceutical comprising thepolymorphic and/or mutant A2M polypeptide or nucleic acid or bindingpartner, or fragment thereof has been obtained, it can be administeredto a organism in need to treat or prevent a neuropathy, such as AD.

[0292] Having now generally described the invention, the followingexamples are offered to illustrate, but not to limit the claimedinvention.

EXAMPLES

[0293] The nucleic acid embodiments of the invention include isolated orpurified nucleic acids comprising, consisting essentially of, orconsisting of an A2M gene (e.g., SEQ ID NO: 1) with one or more of theSNPs and/or mutations described in Table 1. Other embodiments includeisolated or purified nucleic acids comprising, consisting essentiallyof, or consisting of an A2M gene having at least one SNP and/or mutationdescribed in Table 1 along with other SNPs, such as those described inTable 2. Still other embodiments relate to isolated or purified nucleicacid fragments of the A2M gene which include at least one of the SNPsdescribed in Table 1. Such fragments can range in length from at least10, at least 15, at least 20, at least 25, at least 30, at least 40, atleast 50, at least 75, at least 100, a least 150, at least 200, at least250, at least 300, at least 400, at least 500, at least 750, at least1000, at least 2500, at least 5000, at least 7500, at least 10,000, atleast 20,000, at least 30,000, at least 40,000, at least 50,000 orgreater than 50,000 nucleotides and include both exons and introns ofthe A2M gene. Isolated or purified nucleic acid fragments of the A2Mgene having at least one SNP and/or mutation described in Table 1 alongwith other SNPs, such as those described in Table 2, are alsocontemplated. Such fragments can range in length from at least 10, atleast 15, at least 20, at least 25, at least 30, at least 40, at least50, at least 75, at least 100, a least 150, at least 200, at least 250,at least 300, at least 400, at least 500, at least 750, at least 1000,at least 2500, at least 5000, at least 7500, at least 10,000, at least20,000, at least 30,000, at least 40,000, at least 50,000 or greaterthan 50,000 nucleotides and include both exons and introns of the A2Mgene. Other embodiments of the present invention include fragments ofthe A2M gene, wherein the fragments contains at least 9, at least 16, orat least 18 consecutive nucleotides of the polymorphic or mutant A2Mgene but including at least one of the SNPs and/or mutations in Table 1.Isolated or purified nucleic acids that are complementary to said A2Mnucleic acids and fragments thereof are also embodiments. Someembodiments also concern genomic DNA, RNA, and cDNA corresponding topolymorphic and/or mutant A2M genes, described herein. Accordingly, insome contexts, the term “polymorphic and/or mutant A2M nucleic acids”refers not only to the full-length polymorphic and/or mutant A2M nucleicacids (e.g., SEQ ID NOs: 1) but also to fragments of these molecules atleast 9, at least 16, or at least 18 nucleotides in length butcontaining at least one of the SNPs and/or mutations identified in Table1, nucleic acids that are complementary to said full-length sequencesand fragments thereof, and genomic DNA, RNA, and cDNA corresponding tosaid sequences.

[0294] The discovery of SNPs and/or mutations in the A2M gene was madewhile analyzing the sequences of the A2M gene obtained from patientssuffering from AD. The approaches used in these experiments is describedin EXAMPLE 1.

Example 1

[0295] Methods of Identifying SNPs and Other Mutations in the A2M GeneThe following protocol that was used to identify the SNPs and/ormutations described herein in patients from the National Intstitute ofMental Health (NIMH) AD Genetics Initiative Sample. However, it will beappreciated that this protocol has general applicability to any humansubject.

[0296] The A2M gene was identified as a candidate gene linked to ADbased both on its known function and available linkage data. Sample setsof DNA showing strong linkage disequilibrium and/or association in theA2M region were chosen for further study.

[0297] The genomic DNA sequence of the A2M gene was obtained as a partof the draft sequence of chromosome 12 from a Human Genome Projectinformation database located at the University of California Santa Cruzavailable at genome.ucsc.edu. The full-length A2M coding sequence (SEQID NO: 2) and A2M protein (SEQ ID NO: 9) sequences were also obtained.The coordinates of publicly available SNPs in the A2M gene were obtainedfrom bio.chip.org. The program SNPer (available at bio.chip.org) wasused to place the publicly available SNPs in relation to the exons ofthe A2M gene. Exon positions generated by SNPer were verified bycomparing the cDNA sequence (SEQ ID NO: 2) to the genomic database atthe NCBI using (Basic Local Alignment Search Tool) BLASTN with thedefault filter (Altschul, et al. (1990) J. Mol. Biol. 215:403-410).Alternatively, the A2M cDNA sequence was queried against the HighThroughput Genomic Sequence (HTGS) database using BLASTN.

[0298] Subsequent to exon verification, specific regions of the A2M genewere selected for sequencing. Regions selected for sequencing were asfollows: (1) a region beginning approximately 1000 base pairs upstreamof the nucleic acid sequence corresponding to the start codon andextending about 150-200 base pairs beyond last nucleotide of the firstexon; (2) a region beginning approximately 150-200 base pairs upstreamof the nucleic acid sequence corresponding to the beginning of the leastexon of the A2M gene and extending about 700 base pairs beyond lastnucleotide of this exon; and (3) a nucleic acid region surrounding eachexon which begins approximately 150-200 base pairs upstream and endsapproximately 150-200 base pairs downstream of each remaining exon.

[0299] Within the selected regions, 500-800 base pair fragments wereamplified by using amplification primers flanking specific regions ofinterest (forward and reverse primers). In general, primers used foramplification ranged from 20 to 24 nucleotides and had an annealingtemperature between 54-60° C. Amplification was performed using about 30ng of human genomic DNA, 5 μmol of each primer, and HotStarTaq Mix(Qiagen). Thermocycling was initiated by heating for 15 minutes at 95°C. followed by 35 cycles of (a) 94° C. for 30 seconds; (b) primerannealing temperature for 45 seconds; and (c) 72° C. for 1 minute. Thecycling was followed by a final 7 minute extension at 72° C. Subsequentto thermocycling, PCR products were purified then quantitated.

[0300] Both strands of each amplified fragment were sequenced usingsequencing primers complementary to a region near the 3′-end of eachstrand. Approximately, 3.2 pmol of sequencing primer and 12 ng ofamplified fragment were added to sequencing buffer including Big DyeTerminator Mix (Applied Biosystems—ABI) according to the manufacturer'sinstructions. Thermocycling included 30 cycles of (a) 96° C. for 10seconds; (b) 50° C. for 5 seconds; and (c) 60° C. for 4 minutes.Reaction products were purified using CentriSep 96 well plates(Princeton Separations) according to manufacturer's instructions. Datawas collected from purified reaction products using an ABI 3700 DNAAnalyzer.

[0301] Using the above amplification and sequencing protocol, severalSNPs and/or mutations were found in the A2M gene, including both exonand intron regions, in individuals having AD. These results are set outin Table 1 herein.

[0302] In view of the fact that the presence of one or more of SNPsand/or mutations in an individual can present a risk that the individualwill acquire AD, it is contemplated that the SNPs and/or mutationsdescribed in Table 1 (i.e., 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2,17i.1, 20e, 20i, 21i, 28i and 30e) can be indicative for altered riskfor AD. As a preliminary evaluation of the risk associated withpossessing one or more of these SNPs, an association analysis infamilies and individuals having AD was performed. That is, thenucleotide identities at the position of one or more of SNPs and/ormutations included in Table 1 (i.e., 6i, 12i.1, 12i.2, 12e, 14e, 14i.1,14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e) in individuals and familieswith AD were determined and tested by both single SNP associationanalyses and haplotype analyses. EXAMPLE 2 describes these experiments.

Example 2

[0303] Association of A2M SNPs and Haplotypes with Alzheimer's Disease

[0304] The polymorphisms listed in Table 1 can be detected frombiological samples provided by families having members afflicted with ADusing the methods described below as well as methods known to thosehaving ordinary skill in the art. Furthermore, association of one ormore polymorphisms listed in Table 1 with an altered risk of AD can bedetermined using the methods described below as well as those describedin U.S. Pat. No. 6,265,546, the disclosure of which is incorporatedherein by reference in its entirety, and those methods known to thosehaving ordinary skill in the relevant art. As described in Example 1,for each of the polymorphisms listed in Table 1, the A2M-1 allelecorresponds to the allele represented in SEQ ID NO: 1. The A2M-2 allelecorresponds to an allele having the polymorphic change (nucleotidesubstitution or mutation) as indicated in column 3 of Table 1 at thesequence position specified in column 2 of Table 1 (the positions andnucleotides affected by each polymorphism and/or mutation are alsoprovided in the Figure).

[0305] To test for a link between the polymorphisms described herein andAD, samples from families having members afflicted with AD were used. Anexample of an appropriate population is the National Institute of MentalHealth (NIMH) Genetics Initiative AD sample, a large sample of affectedsibling pairs and other small families with AD. It should be noted,however, that any population of families having members meeting thecriteria described below can be used for association and haplotypeanalyses.

[0306] Participants in the NIMH sample were recruited from local memorydisorder clinics, nursing homes, and the surrounding communities withthe only requirement for inclusion in the sample being that each familymember include at least two living blood relatives with memory problems.They were evaluated following a standardized protocol (Blacker, D., etal., Arch. Neurol. 51:1198-1204 (1994)) to assure that they metNINCDS/ADRDA criteria for Probable AD (or in the case of secondaryprobands, Possible AD) (McKhann, G., et al., Neurology 34:939-944(1984)), or research pathological criteria for Definite AD(Khachaturian, Z., Arch. Neurol. 42:1005 (1985)). Among the affectedindividuals, 142 (22.2%) had autopsy confirmation of the diagnosis ofAD. Unaffected relatives, generally siblings, were included when theywere available and willing to participate.

[0307] There were a total of 239 unaffected subjects from 131 families(45.6%). An additional 22 study subjects with blood available who hadunclear phenotypes were considered phenotype unknown, as were 5unaffected subjects with unknown ages, and 19 unaffected subjects below50 years of age (primarily children of affected participants). Therewere a total of 639 individuals affected with AD, from 286 families. Themajority of the affected individuals were sibling pairs (202 families,71%), but there were 46 larger sibships (16%), and 38 families withother structures (13%; e.g., parent-child, first cousin, avuncular,extended). All subjects (or, for significantly cognitively impairedindividuals, their legal guardian or caregiver with power of attorney)gave informed consent.

[0308] The full NIMH sample can be used in the descriptive statisticsfor genotype counts and allele frequencies, for the analyses of age ofonset in affected individuals, and for all of the genetic linkageanalyses (except ASPEX, which uses sibships only). However, because theMantel-Haenzel test, conditional logistic regression, and SibshipDisequalibrium Test and EV-FBAT depend on comparisons of closely relatedaffected and unaffected individuals, they are performed on a subsampleincluding all families in which there is at least one affected and atleast one unaffected sibling with A2M data available: 104 families with217 affected and 181 unaffected siblings.

[0309] In order to avoid examining very early onset AD, which appears tohave a distinct genetic etiology (Blacker, D. & Tanzi, R. E., ArchNeurol 55:294-296 (1998)), only those families in which all examinedaffected individuals experienced the onset of AD at age 50 of later areincluded. Although Late Onset Alzheimer's Disease (LOAD) isconventionally identified based on onset after age 60, families withonsets between 50 and 60 are included because onset in this decade isonly partly explained by the known AD genes. Age of onset is determinedbased on an interview with a knowledgeable informant and review ofmedical records.

[0310] The polymorphisms described herein can be manually genotypedaccording to, for example, the protocol described in Matthijs et al.(Matthijs, G., & Marynen, P., Nuc. Acid Res. 19:5102 (1991)).Alternatively, an appropriate fragment of the A2M gene corresponding tothe region of a polymorphism and/or mutation described herein isamplified and sequenced using the methods described in Example I.

[0311] In one example, manual genotyping is carried out using a 96-wellmicrotiter dish format as follows. Three to 10 nanograms of human DNA ismixed with a reaction buffer, deoxynucleotide mix (e.g. for apoly-[dGdT]STR, the final concentration is 200 mM each of dATP, dCTP,and dTTP; and 2 mM dGTP), 1 mCi alpha-32PdGTP or ³³P-dGTP, 15 pM of eachflanking primer and 0.25 units of Taq polymerase in a total volume of 10μL. The reaction are denatured at 94° C. for 4 minutes, followed by25-30 cycles of 1 minute denaturing at 94° C., 0.5-1 minute annealing(variable temperature, usually 55-65° C.) and extension for 1 minute at72° C. Forty-eight (48) experimental and two control (forstandardization of size) samples are loaded on a gel at one time,thereby increasing the amount of information per gel. Whenever possible(e.g., if maker background is sufficiently low) multiple markers (two tofour markers) are multiplexed, or are temporally staggered (30-45minutes) two to three mm on a single gel. Allele sizes for CEPHindividuals 1331-01 and 1331-02 are used as standards. In the rare eventthat no standards are available for a marker, an initial gel is run,which includes a sequencing ladder, to determine allele sizes in theseindividuals. Two μL of sample are mixed with loading dye andsize-fractionated on a 6% denaturing polyacrylamide gel. The gels arethen dried and placed on X-ray film for 2-24 hrs. at -80° C. and read bytwo independent readers.

[0312] It will be apprciated that the manual geneotyping methoddescribed above is only one method that is available for detectingspecific alleles at polymorphic loci. Several other methods that areuseful for detecting specific alleles at polymorphic loci, in particularhuman polymorphic loci. The preferred method for detecting a particularpolymorphism, depends on the nature of the polymorphism. Several methodsof determining the presence or absence of allelic variants of a gene areprovided below. Methods that are useful are not limited to thosedescribed below, but include all available methods.

[0313] Generally, these methods are based in sequence-specificpolynucleotides, oligonucleotides, probes and primers. Any method knownto those of skill in the art for detecting a specific nucleotide withina nucleic acid sequence or for determining the identity of a specificnucleotide in a nucleic acid sequence is applicable to the methods ofdetermining the presence or absence of an allelic variant of these geneson chromosome 12. Such methods include, but are not limited to,techniques utilizing nucleic acid hybridization of sequence-specificprobes, nucleic acid sequencing, selective amplification, analysis ofrestriction enzyme digests of the nucleic acid, cleavage of mismatchedheteroduplexes of nucleic acid and probe, alterations of electrophoreticmobility, primer specific extension, oligonucleotide ligation assay andsingle-stranded conformation polymorphism analysis. In particular,primer extension reactions that specifically terminate by incorporatinga dideoxynucleotide are useful for detection. Several such generalnucleic acid detection assays are known (see, e.g., U.S. Pat. No.6,030,778).

[0314] Any cell type or tissue may be utilized to obtain nucleic acidsamples, e.g., bodily fluid such as blood or saliva, dry samples such ashair or skin.

[0315] a. Primer Extension-Based Methods

[0316] Several primer extension-based methods for determining theidentity of a particular nucleotide in a nucleic acid sequence have beenreported (see, e.g., PCT Application Nos. PCT/US96/03651 (WO96/29431),PCT/US97/20444 (WO 98/20166), PCT/US97/20194 (WO 98/20019),PCT/US91/00046 (WO91/13075), and U.S. Pat. Nos. 5,547,835, 5,605,798,5,622,824, 5,691,141, 5,872,003, 5,851,765, 5,856,092, 5,900,481,6,043,031, 6,133,436 and 6,197,498.) In general, a primer is preparedthat specifically hybridizes adjacent to a polymorphic site in aparticular nucleic acid molecule. The primer is then extended in thepresence of one or more dideoxynucleotides, typically with at least oneof the dideoxynucleotides being the complement of the nucleotide that ispolymorphic at the site. The primer and/or the dideoxynucleotides may belabeled to facilitate a determination of primer extension and identityof the extended nucleotide.

[0317] A preferred method of genotyping or determining the presence ofan allelic variant two-dye fluorescence polarization detected singlebase extension (FP-SBE (12)) on an LJL-Biosystems Criterion Analyst AD(Molecular Devices, Sunnyvale, Calif.). PCR primers are designed toyield products between 200-400 bp in length, and are used at a finalconcentration of 100-300 nM (Invitrogen Corp., Carlsbad, Calif.) alongwith Taq polymerase (0.25 U/reaction; Qiagen, Valencia, Calif. andRoche, Indianapolis, Ind.) and dNTPs (2.5 uM/rxn; Amersham-Pharmacia,Piscataway, N.J.). All PCR reactions are performed from −10 ng of DNA.General PCR thermo-cycling conditions are as follows: initialdenaturation 3 minutes at 94EC, followed by 30-35 cycles of denaturationat 94EC for 45 seconds, primer-specific annealing temperature (seebelow) for 45 seconds, and product extension at 72EC for 1 minute. Finalextension at 72EC for six minutes. PCR products can be visualized on 2%agarose-gels to confirm a single product of the correct size. PCRprimers and unincorporated dNTPs can be degraded by adding exonuclease I(Exol, 0.1-0.15 U/reaction; New England Biolabs, Beverly, Mass.) andshrimp alkaline phosphatase (SAP, 1U/reaction; Roche, Indianapolis,Ind.) to the PCR reactions and incubating for 1 hour at 37EC, followedby 15 minutes at 95EC to inactivate the enzymes. The single baseextension step is performed by directly adding SBE primer (100 nM;Invitrogen Corp., Carlsbad, Calif.), Thermosequenase (0.4 U/reaction;Amersham-Pharmacia, Piscataway, N.J.), and the appropriate mixture ofR110-ddNTP, TAMRA-ddNTP (3 uM; NEN, Boston, Mass.), and all fourunlabeled ddNTPs (22 or 25 uM; Amersham-Pharmacia, Piscataway, N.J.) tothe Exol/SAP treated PCR product. Acycloprime-FP SNP detection kits(G/A)(Perkin-Elmer, Boston, Mass.) may also be used for the SBEreaction. Incorporation of the SNP specific fluorescent ddNTP isachieved by subjecting samples to 35 cycles of 94EC for 15 seconds and55EC for 30 seconds. The length of the SBE primers are designed to yielda melting temperature T_(m) of 62-64EC. Fluorescent ddNTP incorporationis detected using the Analyst™ AD System (Molecular Devices, Sunnyvale,Calif.) and measuring fluorescent polarization for R110 (excitation at490 nm, emission at 520 nm) and TAMRA (excitation at 550 nm, emission at580 nm). Genotypes are called manually or automatically using themanufacturer's software (‘Allelecaller vers. 1.0’, Molecular Devices,Sunnyvale, Calif.). In view of the polymorphic regions provided herein,SNP specific PCR primers (5′ to 3′ sequences), annealing temperature,product length, SBE primer sequence, SNP location and reference sequenceposition, can readily be determined by those of skill in the art usingwell-known methods.

[0318] b. Polymorphism-Specific Probe Hybridization

[0319] Another detection method is allele specific hybridization usingprobes overlapping the polymorphic site and having about 5, 10, 15, 20,25, or 30 nucleotides around the polymorphic region. The probes cancontain naturally occurring or modified nucleotides (see U.S. Pat. No.6,156,501). For example, oligonucleotide probes may be prepared in whichthe known polymorphic nucleotide is placed centrally (allele-specificprobes) and then hybridized to target DNA under conditions which permithybridization only if a perfect match is found (Saiki et al. (1986)Nature 324:163; Saiki et al. (1989) Proc. Natl. Acad. Sci U.S.A.86:6230; and Wallace et al. (1979) Nucl. Acids Res. 6:3543). Such allelespecific oligonucleotide hybridization techniques may be used for thesimultaneous detection of several nucleotide changes in differentpolymorphic regions. For example, oligonucleotides having nucleotidesequences of specific allelic variants are attached to a hybridizingmembrane and this membrane is then hybridized with labeled samplenucleic acid. Analysis of the hybridization signal will then reveal theidentity of the nucleotides of the sample nucleic acid. In a preferredembodiment, several probes capable of hybridizing specifically toallelic variants are attached to a solid phase support, e.g., a “chip”.Oligonucleotides can be bound to a solid support by a variety ofprocesses, including lithography. For example a chip can hold up to250,000 oligonucleotides (GeneChip, Affymetrix, Santa Clara, Calif.).Mutation detection analysis using these chips comprisingoligonucleotides, also termed “DNA probe arrays” is described e.g., inCronin et al. (1996) Human Mutation 7:244 and in Kozal et al. (1996)Nature Medicine 2:753. In one embodiment, a chip includes all theallelic variants of at least one polymorphic region of a gene. The solidphase support is then contacted with a test nucleic acid andhybridization to the specific probes is detected. Accordingly, theidentity of numerous allelic variants of one or more genes can beidentified in a simple hybridization experiment.

[0320] C. Nucleic Acid Amplification-Based Methods

[0321] In other detection methods, it is necessary to first amplify atleast a portion of a gene prior to identifying the allelic variant.Amplification can be performed, e.g., by PCR and/or LCR, according tomethods known in the art. In one embodiment, genomic DNA of a cell isexposed to two PCR primers and amplification is performed for a numberof cycles sufficient to produce the required amount of amplified DNA. Inanother embodiment, the primers are located between 150 and 350 basepairs apart.

[0322] Alternative amplification methods include: self sustainedsequence replication (Guatelli, J. C. et al. (1990) Proc. Natl. Acad.Sci. U.S.A. 87:1874-1878), transcriptional amplification system (Kwoh,D. Y. et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:1173-1177), Q-BetaReplicase (Lizardi, P. M. et al. (1988) Bio/Technology 6:1197), or anyother nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques well known to those of skill inthe art. These detection schemes are especially useful for the detectionof nucleic acid molecules if such molecules are present in very lownumbers.

[0323] Alternatively, allele specific amplification technology, whichdepends on selective PCR amplification may be used in conjunction withthe alleles provided herein. Oligonucleotides used as primers forspecific amplification may carry the allelic variant of interest in thecenter of the molecule (so that amplification depends on differentialhybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) orat the extreme 3′ end of one primer where, under appropriate conditions,mismatch can prevent, or reduce polymerase extension (Prossner (1993)Tibtech 11:238; Newton et al. (1989) Nucl. Acids Res. 17:2503). Inaddition it may be desirable to introduce a restriction site in theregion of the mutation to create cleavage-based detection (Gasparini etal. (1992) Mol. Cell Probes 6:1).

[0324] d. Nucleic Acid Sequencing-Based Methods

[0325] Any of a variety of sequencing reactions known in the art can beused to directly sequence at least a portion of a gene and to detectallelic variants, e.g., mutations, by comparing the sequence of thesample sequence with the corresponding wild-type (control) sequence.Exemplary sequencing reactions include those based on techniquesdeveloped by Maxam and Gilbert (1977) Proc. Natl. Acad. Sci. U.S.A.74:560) or Sanger et al. (1977) Proc. Natl. Acad. Sci 74:5463. It isalso contemplated that any of a variety of automated sequencingprocedures may be used when performing the subject assays ((1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,for example, U.S. Pat. Nos. 5,547,835, 5,691,141, and International PCTApplication No. PCT/US94/00193 (WO 94/16101), entitled “DNA Sequencingby Mass Spectrometry” by H. Koster; U.S. Pat. Nos. 5,547,835, 5,622,824,5,851,765, 5,872,003, 6,074,823, 6,140,053 and International PCTApplication No. PCT/US94/02938 (WO 94/21822), entitled “DNA Sequencingby Mass Spectrometry Via Exonuclease Degradation” by H. Koster, and U.S.Pat. Nos. 5,605,798, 6,043,031, 6,197,498, and International PatentApplication No. PCT/US96/03651 (WO 96/29431) entitled “DNA DiagnosticsBased on Mass Spectrometry” by H. Koster; Cohen et al. (1996) AdvChromatogr 36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol38:147-159). It will be evident to one skilled in the art that, forcertain embodiments, the occurrence of only one, two or three of thenucleic acid bases need be determined in the sequencing reaction. Forinstance, A-track sequencing or an equivalent, e.g., where only onenucleotide is detected, can be carried out. Other sequencing methods areknown (see, e.g., in U.S. Pat. No. 5,580,732 entitled “Method of DNAsequencing employing a mixed DNA-polymer chain probe” and U.S. Pat. No.5,571,676 entitled “Method for mismatch-directed in vitro DNAsequencing”).

[0326] e. Restriction Enzyme Digest Analysis

[0327] In some cases, the presence of a specific allele in nucleic acid,particularly DNA, from a subject can be shown by restriction enzymeanalysis. For example, a specific nucleotide polymorphism can result ina nucleotide sequence containing a restriction site which is absent fromthe nucleotide sequence of another allelic variant.

[0328] f. Mismatch Cleavage

[0329] Protection from cleavage agents, such as, but not limited to, anuclease, hydroxylamine or osmium tetroxide and with piperidine, can beused to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, thetechnique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing a control nucleic acid, which is optionallylabeled, e.g., RNA or DNA, comprising a nucleotide sequence of anallelic variant with a sample nucleic acid, e.g, RNA or DNA, obtainedfrom a tissue sample. The double-stranded duplexes are treated with anagent, which cleaves single-stranded regions of the duplex such asduplexes formed based on basepair mismatches between the control andsample strands. For instance, RNA/DNA duplexes can be treated with RNaseand DNA/DNA hybrids treated with S1 nuclease to enzymatically digest themismatched regions.

[0330] In other embodiments, either DNA/DNA or RNA/DNA duplexes can betreated with hydroxylamine or osmium tetroxide and with piperidine inorder to digest mismatched regions. After digestion of the mismatchedregions, the resulting material is then separated by size on denaturingpolyacrylamide gels to determine whether the control and sample nucleicacids have an identical nucleotide sequence or in which nucleotides theydiffer (see, for example, Cotton et al. (1988) Proc. Natl Acad SciU.S.A. 85:4397; Saleeba et al. (1992) Methods Enzymod. 217:286-295). Thecontrol or sample nucleic acid is labeled for detection.

[0331] g. Electrophoretic Mobility Alterations

[0332] In other embodiments, alteration in electrophoretic mobility isused to identify the type of allelic variant of a gene of interest. Forexample, single-strand conformation polymorphism (SSCP) may be used todetect differences in electrophoretic mobility between mutant and wildtype nucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci. U.S.A.86:2766, see also Cotton (1993) Mutat Res 285:125-144; and Hayashi(1992) Genet Anal Tech Appl 9:73-79). Single-stranded DNA fragments ofsample and control nucleic acids are denatured and allowed to renature.The secondary structure of single-stranded nucleic acids variesaccording to sequence, the resulting alteration in electrophoreticmobility enables the detection of even a single base change. The DNAfragments may be labeled or detected with labeled probes. Thesensitivity of the assay may be enhanced by using RNA (rather than DNA),in which the secondary structure is more sensitive to a change insequence. In another embodiment, the subject method uses heteroduplexanalysis to separate double stranded heteroduplex molecules on the basisof changes in electrophoretic mobility (Keen et al. (1991) Trends Genet7:5).

[0333] h. Polyacrylamide Gel Electrophoresis

[0334] In yet another embodiment, the identity of an allelic variant ofa polymorphic region of an gene is obtained by analyzing the movement ofa nucleic acid comprising the polymorphic region in polyacrylamide gelscontaining a gradient of denaturant is assayed using denaturing gradientgel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). WhenDGGE is used as the method of analysis, DNA will be modified to ensurethat it does not completely denature, for example by adding a GC clampof approximately 40 bp of high-melting GC-rich DNA by PCR. In a furtherembodiment, a temperature gradient is used in place of a denaturingagent gradient to identify differences in the mobility of control andsample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:1275).

[0335] i. Oligonucleotide Ligation Assay (OLA)

[0336] In another embodiment, identification of the allelic variant iscarried out using an oligonucleotide ligation assay (OLA), as described,e.g., in U.S. Pat. No. 4,998,617 and in Landegren, U. et al. (1988)Science 241:1077-1080. The OLA protocol uses two oligonucleotides whichare designed to be capable of hybridizing to abutting sequences of asingle strand of a target. One of the oligonucleotides is linked to aseparation marker, e.g,. biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al. (1990)Proc. Natl. Acad. Sci. U.S.A. 87:8923-8927). In this method, PCR is usedto achieve the exponential amplification of target DNA, which is thendetected using OLA.

[0337] Several techniques based on this OLA method have been developedand can be used to detect specific allelic variants of a polymorphicregion of a gene. For example, U.S. Pat. No. 5,593,826 discloses an OLAusing an oligonucleotide having 3′-amino group and a 5′-phosphorylatedoligonucleotide to form a conjugate having a phosphoramidate linkage. Inanother variation of OLA described in Tobe et al. (1996) Nucl. AcidsRes. 24:3728, OLA combined with PCR permits typing of two alleles in asingle microtiter well. By marking each of the allele-specific primerswith a unique hapten, i.e. digoxigenin and fluorescein, each OLAreaction can be detected by using hapten specific antibodies that arelabeled with different enzyme reporters, alkaline phosphatase orhorseradish peroxidase. This system permits the detection of the twoalleles using a high throughput format that leads to the production oftwo different colors.

[0338] j. SNP Detection Methods

[0339] Several methods have been developed to facilitate the analysis ofsingle nucleotide polymorphisms.

[0340] In one embodiment, the single base polymorphism can be detectedby using a specialized exonuclease-resistant nucleotide, as disclosed,e.g., in Mundy, C. R. (U.S. Pat. No. 4,656,127). According to themethod, a primer complementary to the allelic sequence immediately 3′ tothe polymorphic site is permitted to hybridize to a target moleculeobtained from a particular animal or human. If the polymorphic site onthe target molecule contains a nucleotide that is complementary to theparticular exonuclease-resistant nucleotide derivative present, thenthat derivative will be incorporated onto the end of the hybridizedprimer. Such incorporation renders the primer resistant to exonuclease,and thereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

[0341] In another embodiment, a solution-based method for determiningthe identity of the nucleotide of a polymorphic site is employed (Cohen,D. et al. (French Patent 2,650,840; PCT Application No. WO91/02087)). Asin the Mundy method of U.S. Pat. No. 4,656,127, a primer is employedthat is complementary to allelic sequences immediately 3′ to apolymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

[0342] k. Genetic Bit Analysis

[0343] An alternative method, known as Genetic Bit Analysis or GBA™ isdescribed by Goelet, et al. (U.S. Pat. No. 6,004,744, PCT ApplicationNo. 92/15712). The method of Goelet, et al. uses mixtures of labeledterminators and a primer that is complementary to the sequence 3′ to apolymorphic site. The labeled terminator that is incorporated is thusdetermined by, and complementary to, the nucleotide present in thepolymorphic site of the target molecule being evaluated. In contrast tothe method of Cohen et al. (French Patent 2,650,840; PCT Application No.WO91/02087), the method of Goelet, et al. is preferably a heterogeneousphase assay, in which the primer or the target molecule is immobilizedto a solid phase.

[0344] l. Other Primer-Guided Nucleotide Incorporation Procedures

[0345] Other primer-guided nucleotide incorporation procedures forassaying polymorphic sites in DNA have been described (Komher, J. S. etal. (1989) Nucl. Acids Res. 17:7779-7784; Sokolov, B. P. (1990) Nucl.Acids Res. 18:3671; Syvanen, A. C., et al. (1990) Genomics 8:684-692,Kuppuswamy, M. N. et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.)88:1143-1147; Prezant, T. R. et al. (1992) Hum. Mutat. 1:159-164;Ugozzoli, L. et al. (1992) GATA 9:107-112; Nyren, P. et al. (1993) Anal.Biochem. 208:171-175). These methods differ from GBATM in that they allrely on the incorporation of labeled deoxynucleotides to discriminatebetween bases at a polymorphic site. In such a format, since the signalis proportional to the number of deoxynucleotides incorporated,polymorphisms that occur in runs of the same nucleotide can result insignals that are proportional to the length of the run (Syvanen, A. C.,et al. (1993) Amer. J. Hum. Genet. 52:46-59).

[0346] For determining the identity of the allelic variant of apolymorphic region located in the coding region of a gene, yet othermethods than those described above can be used. For example,identification of an allelic variant which encodes a mutated protein canbe performed by using an antibody specifically recognizing the mutantprotein in, e.g., immunohistochemistry or immunoprecipitation. Bindingassays are known in the art and involve, e.g., obtaining cells from asubject, and performing binding experiments with a labeled lipid, todetermine whether binding to the mutated form of the protein differsfrom binding to the wild-type protein.

[0347] m. Molecular Structure Determination

[0348] If a polymorphic region is located in an exon, either in a codingor non-coding region of the gene, the identity of the allelic variantcan be determined by determining the molecular structure of the mRNA,pre-mRNA, or cDNA. The molecular structure can be determined using anyof the above described methods for determining the molecular structureof the genomic DNA, e.g., sequencing and single-strand conformationpolymorphism.

[0349] n. Mass Spectrometric Methods

[0350] Nucleic acids can also be analyzed by detection methods andprotocols, particularly those that rely on mass spectrometry (see, e.g.,U.S. Pat. Nos. 5,605,798, 6,043,031, 6,197,498, and International PatentApplication No. WO 96/29431, International PCT Application No. WO98/20019).

[0351] Multiplex methods allow for the simultaneous detection of morethan one polymorphic region in a particular gene. This is the preferredmethod for carrying out haplotype analysis of allelic variants of agene.

[0352] Multiplexing can be achieved by several different methodologies.For example, several mutations can be simultaneously detected on onetarget sequence by employing corresponding detector (probe) molecules(e.g., oligonucleotides or oligonucleotide mimetics). Variations inadditions to those set forth herein will be apparent to the skilledartisan.

[0353] A different multiplex, detection format is one in whichdifferentiation is accomplished by employing different specific capturesequences which are position-specifically immobilized on a flat surface(e.g., a ‘chip array’).

[0354] o. Other Methods

[0355] Additional methods of analyzing nucleic acids includeamplification-based methods including polymerase chain reaction (PCR),ligase chain reaction (LCR), mini-PCR, rolling circle amplification,autocatalytic methods, such as those using QJ replicase, TAS, 3SR, andany other suitable method known to those of skill in the art.

[0356] Other methods for analysis and identification and detection ofpolymorphisms, include but are not limited to, allele specific probes,Southern analyses, and other such analyses.

[0357] Five groups of statistical analyses can be used to explore therelationship between A2M and AD in study families. First, the A2Mgenotype and allele frequencies for affected and unaffected individualsare calculated. Second, stratified on families, Mantel-Haenzel oddsratios (see Mantel, H. & Haenszel, W. J. Natl. Cancer Inst. 22:719-748(1959), the disclosure of which is incorporated by reference in itsentirety) are calculated for the effect of possessing an allele for eachpolymorphism and/or mutation described herein on altering the risk forAD, and conditional logistic regression, conditioning on family, is usedto control for the effect of APOE-ε4. Third, association for eachpolymorphism and/or mutation described herein is tested for using theSibship Disequilibrium Test (SDT) of Horvath and Laird (Horvath, S. &Laird, N., Am. J. Hum. Genet. 63:1886-1897 (1998), the disclosure ofwhich is incorporated by reference in its entirety), a variation of theTransmission Disequilibrium Test (TDT) that is able to detect linkageand association in the absence of parental data or the FBAT or EV-FBATdeveloped by Rabinowitz and Laird (Rabinowitz, D & Laird, N., Hum.Hered. 50:211-23 (2000), the disclosure of which is incorporated byreference in its entirety). Fourth, a variety of techniques are used toassess whether any A2M effect occurs via a change in age of onset.Fifth, several genetic association methods can be used to assess therelationship between A2M and AD, and whether any allelic associationmight be related to the recent report of linkage to centromeric markerson chromosome 12. Wherever possible, APOE-ε4 effects are controlled forby stratification or by including APOE-ε4 as a covariate in multivariateanalyses. Except as otherwise noted, the analyses reported here can beperformed using statistical analysis software such as, the SASstatistical analysis package (SAS Institute, SAS Program Guide, Version6, Cary, N.C. (1989)).

[0358] For all types of analysis, allele frequencies are computed fromthe data, but rare alleles can be adjusted up to a frequency of 0.01(with a compensatory small decrease in the frequency of the most commonalleles) in order to minimize the possibility of a false positiveresult. All analyses are repeated using the uncorrected frequencies.

[0359] For descriptive purposes, A2M genotype counts and allelefrequencies are examined in affected and unaffected subjects in studyfamilies. Unaffected individuals in AD families are not geneticallyindependent of their affected relatives, of course, and thus would beexpected to show higher frequencies of AD-associated alleles compared tothe general population. However, given an increased risk of AD with agiven allele, its frequencies would be expected to be higher amongaffected individuals than among their unaffected relatives. However,since these frequencies are pooled across families, they are neither asaccurate nor as powerful an indicator of genetic association as the SDT.

[0360] A2M genotype counts and allele frequencies for each polymorphismdescribed herein are reported separately for primary and secondaryprobands, with primary probands serving as the primary subjectpopulation, and secondary probands as a confirmation sample. Allelefrequencies in the probands are compared to those for unaffectedindividuals based on the oldest unaffected individuals from each of the105 families in which one or more unaffected subjects with A2M data isavailable. In addition, the analyses are repeated using an unaffectedsample that had passed through a majority of the age of risk, the“stringent” unaffecteds, those who are at least as old as the age ofonset of the latest-onsetting affected family member, again selectingthe oldest such individual in each family. Because age of onset iscorrelated in families (Farrer, L. A., et al., Neurology 40:395-403(1990)), using onset ages in the subjects' own families is preferable tosetting an arbitrary cutoff.

[0361] Initial genotype counts and allele frequencies for eachpolymorphism and/or mutation described herein are determined (Matthijs,G., Marynen, P., Nuc. Acid. Res. 19:5102 (1991)) in primary probands,secondary probands, unaffected individuals (oldest in family), and“stringent” unafffecteds, (those who have reached the onset age of thelatest-onsetting affected, again using the oldest such individual),stratified on individual APOE dose.

[0362] Mantel-Haenzel odds ratios (see Mantel, H. & Haenszel, W. J.Natl. Cancer Inst. 22:719-748 (1959), the disclosure of which isincorporated by reference in its entirety) can be calculated for theodds of being affected given the possession of at least one allele of apolymorphism described herein. These analyses are preformed stratifiedon family using n-to-m matching, so all members of a sibship can be usedand intercorrelations among siblings can be taken into account. Spielmanand Ewens (Spielman, R. S., and Ewens, W. J. Am. J. Hum. Genet.62:450-458 (1998)) have suggested the use of a similar analysis to testfor linkage. The analyses are performed first using all unaffectedsiblings, and then only the stringent unaffected siblings.

[0363] Conditional logistic regression is used to control theMantel-Haenzel odds ratio for the effect of APOE-ε4 on AD risk. Here,the outcome is disease status of each sibling, conditioning on familyusing an n-to-m matching paradigm, and including APOE-ε4/ε4 homozygosityas a covariate, along with a term for the interaction between APOE-ε4and A2M alleles of polymorphisms described herein. Like theMantel-Haenzel odds ratio, conditional logistic regression is a standardmethod for analysis of data from matched sets, and can control forclustering of genotypes within families of arbitrary size. Theseanalyses are performed using the PHREG procedure in SAS (SAS Institute,SAS Program Guide, Version 6, Cary N.C. (1989)). These analyses arerepeated using only the “stringent” unaffected siblings (those who wereas least as old as the onset age of the oldest-onsetting affectedsibling) in order to minimize the effect of misclassification ofunaffected siblings. These analyses can also be performed coding APOE-ε4as gene dosage, and including a term for the possession of an APOE-2allele, previously shown to decrease disease risk (Corder, E. H., etal., Nat. Genet. 7:180-184 (1994); Farrer, L. A., et al. JAMA278;1349-1356 (1997)).

[0364] Mantel-Haenzel odds ratios and p-values for the association ofA2M alleles for each polymorphism described herein with risk of AD willbe greater than 2 and less than 0.05, respectively. Conditional logisticregression analyses, which allow for the calculation of Mantel-Haenzelodds ratios adjusted for the effect of APOE-ε4 on AD risk, are alsoexpected to generate statistically significant p-values (less than 0.05)for association of A2M alleles for each polymorphism described hereinwith risk of AD. Interaction between A2M alleles for each polymorphismdescribed herein and APOE-ε4 are not expected to be statisticallysignificant.

[0365] The Sibship Disequilibrium Test (SDT) (Horvath, S. & Laird, N.,Am. J. Hum. Genet. 63:1886-1897 (1998), the disclosure of which isincorporated by reference in its entirety) is a non-parametric sign testdeveloped for use with sibling pedigree data that compares the averagenumber of candidate alleles between affected and unaffected siblings.The SDT is similar to the S-TDT, a recently developed test that alsodoes not require parental data (Spielman, R. S., and Ewens, W. J., Am.J. Hum. Genet. (Suppl.) 53:363 (1993) the disclosure of which isincorporated herein by reference in its entirety), but has the advantageof being able to detect association in sibships of an arbitrary size.Like the TDT, S-TDT, and other family-based association tests, the SDToffers the advantage of not being susceptible to errors due toadmixture. Another advantage of these methods is that misclassificationof affection status (e.g., due to the unaffected siblings not havingpassed through the age of risk) decreases the power of the test, butdoes not lead to invalid results. The SDT can test for both linkage andlinkage disequilibrium; it can only detect linkage disequilibrium in thepresence of linkage, hence there is no confounding due to admixture. Thenull hypothesis of the SDT is that Θ=½ (no linkage) or δ=0 (nodisequilibrium), i.e., H₀:δ(Θ)−½)=0. The SDT program (for severalplatforms) and documentation may be found atftp://sph70-57.harvard.edu/XDT/.

[0366] Because the SDT does not require parental data, and can use allinformation from sibships of arbitrary size, it is well-suited to theanalysis of the NIMH AD data. Before using it to detect novel AD genes,the SDT is validated with the known AD gene APOE-ε4 in the sample. Forexample, in an examination of 150 sibships with 286 affected and 242unaffected individuals from the sample, the SDT was able to detect notonly the deleterious APOE-ε4 effect but also the more difficult todetect APOE-2 protective effect (Farrer, L. A., et al., JAMA278:1349-1356 (1997); Corder, E. H., et al., Nature Genet. 7:180-184(1994)) not previously detected in these data (Blacker, D., et al.,Neurology 48:139-147 (1997)).

[0367] The primary analysis of the association of A2M polymorphisms withAD examines the probability of passing along an A2M polymorphic alleleas a function of affection status. In order to increase the likelihoodof correct classification of unaffected status, the analyses arerepeated including only “stringent” unaffected siblings, those who wereat least as old as the latest on setting affected siblings, a sample of60 families. In addition, in order to assess whether the effect differedin different APOE genotypes persists in individuals with similar APOEgenotypes, the analyses are repeated within strata defined by matchingaffected and unaffected siblings for APOE-ε4 gene dose. To providefurther validation of the SDT, the Sibling TDT (Spielman, R. S. andEwens, W. J., Am. J. Hum. Genet. 62:450-458 (1998), the disclosure ofwhich is incorporated herein by reference in its entirety) (S-TDT) isapplied.

[0368] The SDT Z values and p-values for the association of A2M allelesfor each polymorphism described herein with risk of AD will be greaterthan 2 and less than 0.05, respectively. The SDT values are expected tobe confirmed by the S-TDT.

[0369] The general approach to family-based examinations described byRabinowitz and Laird (Rabinowitz, D & Laird, N., Hum. Hered. 50:211-23(2000), the disclosure of which is incorporated by reference in itsentirety) (FBAT and EV-FBAT) can also be used to test the associationbetween the A2M alleles of the polymorphisms described herein and riskof AD. This approach is based on computing p-values by comparing teststatistics for association to their conditional distributions given theminimal sufficient statistic under the null hypothesis for the geneticmodel, sampling plan and population admixture. The approach can beapplied with any test statistic, so any kind of phenotype andmulti-allelic markers may be examined, and covariates may be included inanalyses. By virtue of the conditioning, the approach results in correcttype I error probabilities regardless of population admixture, the truegenetic model and the sampling strategy. The EV-FBAT test statistics andp-values for the association of A2M alleles for each polymorphismdescribed herein with risk of AD will be greater than 2 and less than0.05, respectively.

[0370] In order to see if A2M effects appear to operate via changes inage of onset, affected individuals are examined according to A2Mgenotype, stratifying on or controlling for the powerful effect ofAPOE-ε4. First, this is examined graphically using Kaplan Meier curvesincluding all affected and unaffected individuals, first stratifying onA2M genotype alone, and then on A2M risk allele carrier status for eachpolymorphism describe herein and APOE-ε4 dose. Second, the mean ages ofonset of primary and secondary probands are compared by A2M genotypeoverall, and stratified on APOE-ε4 gene dose. Third, analysis ofvariance (performed separately for primary and secondary probands) isused, including first only A2M genotype (defined as any 2 vs. none),then only APOE genotype (defined as APOE-ε4 gene dose or APOE-ε4/ε4 vs.not), then both, and then both plus an interaction term.

[0371] Analyses of haplotypes that are associated with AD can beperformed using software such as TRANSMIT version 2.5 (Clayton, (1999)Am. J. Hum. Genet. 65: 1170-1177, see also Clayton et al., (1999) Am. J.Hum. Genet. 65: 1161-1169, the disclosures of which are incorporatedherein by reference in their entireties). This approach is ageneralization of the TDT and uses an expectation-maximization (EM)algorithm to reconstruct haplotypes with missing parental genotypes.Nominal global p-values are estimated using the empirical variancefunction.

[0372] For all types of analyses, allele frequencies are computed fromthe data, but rare alleles are adjusted up to a frequency of 0.01 (witha compensatory small decrease in the frequency of the most commonalleles) in order to minimize the possibility of a false positiveresult. All analyses are repeated using the uncorrected frequencies.

[0373] The association analysis and haplotype analysis can be performedfor the SNPs and/or mutations described herein using the methodologyemployed in U.S. Pat. Nos. 6,265,546; 6,090,620; 6,201,107; or6,303,307; all of which are hereby expressly incorporated by referencein their entireties. The p-values for the association of haplotypes,which include A2M alleles for polymorphism and/or mutations describedherein, with risk of AD will be less than 0.05.

[0374] SNP 18i (the site of a five base pair deletion of the sequenceACCAT located 1 base pair upstream of exon 18, see the Figure) and 24epolymorphism (site of a nucleotide substitution of A to G at nucleotideposition 145 within exon 24 which results in an isoleucine to valinesubstitution in the A2M polypeptide (SEQ ID NO: 9) at amino acidposition 1000, see the Figure) were examined for association with ADusing some of the above-described methods. Specifically, the Sibling TDTdescribed by Spielman and Ewens and the EV-FBAT described by Rabinowitzand Laird were determined. For 18i the population sample size was 76 andfor 24e the sample size was 110. The p-value for the association of the18i deletion with AD was 0.0002 using EVA-BAT and 0.0015 using S-TDTwhereas the p-value for the association of the 24e polymorphism with ADwas 0.09 using EV-FBAT and 0.14 using S-TDT. Accordingly, the A2M-2allele of 18i showed strong statistical significance for associationwith AD and the A2M-2 allele of 24e displayed a trend for association.

[0375] The 21i polymorphism described herein was tested for associationwith AD using the Sibling TDT and EV-FBAT as above. The population thatwas sampled has an effective size of 92 individuals. The frequency ofthe minor allele in this population was 0.22. The p-value calculatedusing the S-TDT was 0.001 whereas the p-value calculated using theEV-FBAT was 0.004. Each of these values are statistically significantand provide evidence that the 21i polymorphism is associated with anincreased risk of incurring AD.

[0376] Table 3 displays the results of similar analyses that wereperformed for 21 i from other sample populations and for other SNPsand/or mutations described in Table 1. In particular, Table 3 lists thesize of the population of AD patients sampled for each SNP and/ormutation and the frequency of the minor allele in that population. Thep-values (based on EV-FBAT statistics) for each of these SNPs and/ormutations samples are also provided in Table 3. In some cases, thepopulation was made up entirely of affected individuals over the age of65. In these cases, a separate p-value is included that represents thesignificance of the association of the examined SNP and/or mutation withthe development of Late Onset AD (LOAD). EVA-BAT-based p-values that areless than or equal to 0.05 indicate statistical significance.Additionally, for each SNP and/or mutation that was investigated, Table3 provides an odds ratio (OR) and the corresponding 95% confidenceinterval, which describes the association with AD for both heterozygousand homozygous genotypes. TABLE 3 Genetic Association of Individual SNPsand/or Mutations with Alzheimer's Disease Odds Ratio (95% Odds Ratio(95% Minor Confidence Confidence SNP/ Sample Allele p-value Interval)for a Interval) for two Mutation Size Frequency (EV-FBAT) single minorallele minor alleles 12e 37 0.06 0.0009 3.62 (1.79, 7.34) 12.9 (0.94,176) 12e 39 0.07 0.0018 3.18 (1.69, 5.99) 11.6 (0.88, 154) 12e  31* 0.070.0031* ND ND 21i 92 0.22 0.004 2.00 (1.34, 3.02) 4.01 (1.27, 11.8) 21i71 0.17 0.041 1.72 (1.16, 2.56) 1.84 (0.55, 6.11) 21i  50* 0.17 0.0039*ND ND

[0377] Haplotype analyses were performed for groups of either five orsix SNPs and/or mutations described in Table 1. The nominal p-value foreach haplotype as calculated using TRANSMIT ver 2.5 is provided below inTable 4. In some cases, the population was made up entirely of affectedindividuals over the age of 65. In these cases, a separate p-value isincluded that represents the significance of the association of theexamined SNP and/or mutation with the development of Late Onset AD(LOAD). Nominal p-values that are less than or equal to 0.05 indicatestatistical significance. TABLE 4 Association of Haplotypes withAlzheimer's Disease Haplotype Nominal p-value 6i, 12e, 14i.1, 18i, 20e0.07 6i, 12e, 14i.1, 18i, 21i 0.0032 6i, 12e, 14i.1, 18i, 21i* 0.06012e, 14i.1, 18i, 21i, 24e 0.0031 12e, 14i.1, 18i, 21i, 24e* 0.033 14i.1,18i, 20e, 21i, 24e 0.040 18i, 20e, 21i, 24e, 28i 0.0016 6i, 12e, 14i.1,18i, 21i, 24e 0.00023 6i, 12e, 14i.1, 18i, 21i, 24e* 0.014

[0378] The results demonstrate that haplotypes that includepolymorphisms of the A2M gene provided herein associate with risk forAD. Furthermore, the results indicate that at least a few of the testedhaplotypes can be associated with an increased risk of LOAD. Thenucleotide identities of the haplotypes are the three most commoncombinations of genotypes as determined in the NIMH sample set using theTRANSMIT analysis program. Thus, in methods provided herein whichinclude genotyping an individual for the polymorphisms included in thehaplotypes, a step can be determining the identity of the nucleotide(s)to see if it is consistent with any of these three most commonhaplotypes.

[0379] It will be appreciated that other haplotypes which include one ormore the SNPs and/or mutations described in Table 1 in combination withSNPs and/or mutations that are described in Table 2 are likely to beimplicated with an increased risk of AD.

Example 3

[0380] Screening Potential Therapeutics by Analyzing Clearance of Aβ byPolymorphic A2M

[0381] The activation of polymorphic and/or mutant A2M (A2M) by Aβ(amyloid β) can be detected by monitoring the LRP-mediated clearance ofAβ. HE 293 cells expressing LRP (LRP:TCRζchimera) are seeded in 384 wellmicroplates and grown in DMEM. HEK 293 cells not expressing LRP(IL-2:TCRζ chimeras) are used as negative controls. To each well isadded 5, 20, 50 or 100 μg of test compound in DMEM. After an hourincubation at 37° C., unlabeled Aβ and polymorphic A2M from the mediaand extracts of the transfected cells are added. Unlabeled Aβ togetherwith wildtype A2M (Sigma) are also tested as a positive control. After 3days, the supernatant is removed from each well and Aβ levels aredetermined by ELISA.

[0382] To monitor the clearance of Aβ by ELISA, each well of themicroplate is blocked with 200 μL of 1% BSA in Tris buffered saline pH7.4 (TBS) for 1 hour. After the incubation, the supernatant is removedand each well is washed three times with 200 μL of TBS containing 0.1%Tween-20. 50 μL of a 1:3000 dilution of Aβ1-12 alkaline phosphataseconjugated monoclonal antibody 436 in TBS containing 1% BSA is added toeach well and the microplate is incubated at room temperature for 1hour. After the incubation, the supernatant is removed and each well iswashed as described above. 50 μL of CDP-Star (Sapphire) luminescencesubstrate is added to each well and the plate is incubated in the darkfor 5 minutes. The luminescence of each well is then quantitated usingan ABI TR717 luminometer.

[0383] Compounds that enhance the binding of Aβ to A2M promote thesubsequent clearance of A2M/Aβ complexes from the medium via LRP.Accordingly, decreased luminescence indicates compounds that enhance thebinding of Aβ to A2M.

Example 4 Screening Potential Therapeutics by Analyzing the Binding ofPolymorphic A2M to Cells Expressing LRP

[0384] To screen for therapeutic compounds capable of modulating thebinding of polymorphic A2M to LRP, A2M from the media and extracts ofthe transfected cells are labeled with ¹²⁵I then treated with 5, 20, 50or 100 μg of test compound in Tris/HCl or sodium phosphate buffer at 37°C. for 2 hours. Untreated polymorphic A2M and wildtype A2M labeled with¹²⁵I are used as controls. A2M can be labeled with ¹²⁵I using kit forradiolabeling proteins obtainable from Pierce according to themanufacturer's instructions.

[0385] HEK 293 cells expressing LRP (LRP:TCR9 chimera) and HEK 293 cellslacking LRP (IL-2:TCRΘ chimeras) are seeded in 96 well microplates andgrown for 18 hours in DMEM. Subsequent to growth, the cells are washedwith 0.2 mL DMEM then pre-incubated for 30 minutes with 0.2 mL of assaymedium comprising DMEM, 1.5% BSA, and 20 mM Hepes at pH 7.4. After thepre-incubation, the assay medium is removed and about 0.1 pmol of the¹²⁵I-labeled A2M samples described above are added to duplicate wells in0.1 mL of assay medium. To control for nonspecific background, wells towhich no cells are added and wells to which no compounds are added arealso included. Additional controls for binding specificity include wellsto which 100-fold excess cold wildtype A2M or cold receptor associatedprotein (RAP) is added. Both RAP and cold wildtype A2M act inhibitors oflabeled A2M binding.

[0386] After a 1 hour incubation at 4° C., the media layer is removedand the cells are washed twice with 1 mL of isotonic phosphate bufferedsaline (PBS). The cell layer is then solubilized using 0.5 mL of 10 NNaOH. The cell-bound ¹²⁵I-labeled A2M is quantified using a gammacounter.

Example 5 Screening Potential Therapeutics by Analyzing theInternalization and Degradation of Polymorphic A2M

[0387] To screen for therapeutic compounds capable of promoting theinternalization and degradation of polymorphic A2M, A2M from the mediaand extracts of the transfected cells are labeled with ¹²⁵I then treatedwith 5, 20, 50 or 100 μg of test compound in Tris/HCl or sodiumphosphate buffer at 37° C. for 2 hours. Untreated polymorphic A2M andwildtype A2M labeled with ¹²⁵I are used as controls. A2M can be labeledwith an ¹²⁵I labeling kit for radiolabeling proteins obtainable fromcommercial suppliers, according to the manufacturer's instructions.

[0388] HEK 293 cells expressing LRP (LRP:TCRΘ chimera) and HEK 293 cellslacking LRP (IL-2:TCRΘ chimeras) are seeded in 48 well microplate andgrown for 10 days in DMEM. Subsequent to growth, the cells are washedwith 1 mL DMEM then pre-incubated for 30 minutes with 0.5 mL of assaymedium comprising DMEM, 1.5% BSA, and 20 mM Hepes at pH 7.4. After thepre-incubation, the assay medium is removed and about 0.1 pmol of the¹²⁵I-labeled A2M samples described above are added to duplicate wells in0.4 mL of assay medium. To control for nonspecific background, wells towhich no cells are added and wells to which no compounds are added arealso included. Additional controls for binding specificity include wellsto which 100-fold excess cold wildtype A2M or cold receptor associatedprotein (RAP) is added. Both RAP and cold wildtype A2M act as inhibitorsof labeled A2M binding.

[0389] After a 2 hour incubation at 37° C., the media layer is removedand added to 50% trichloroacetic acid (TCA). The nondegraded material inthe sample is precipitated by centrifugation at 14,000 g. The amount ofdegraded material present in each sample is determined by counting 0.3mL using a gamma counter. The cell layer is washed twice with 1 mL ofisotonic phosphate buffered saline (PBS). The cell layer is thensolubilized using 0.3 mL of 10 N NaOH. This layer represents thecell-bound and internalized ¹²⁵I-labeled A2M is quantified using a gammacounter.

Example 6 Screening Potential Therapeutics by Analyzing Aβ Binding ofPolymorphic A2M

[0390] To screen for therapeutic compounds capable of modulating theability of polymorphic A2M to bind Aβ, A2M from the media and extractsof the transfected cells are treated with 5, 20, 50 or 100 μg of testcompound in Tris/HCl or sodium phosphate buffer at 37° C. for 2 hours.Untreated A2M and untreated A2M that has been activated with methylamineare used as controls.

[0391] One method of detecting the binding of Aβ to A2M is through anassay based on gel-filtration chromatography. A second method is byimmunoblot analysis. Both of these methods have been used successfullyby other investigators to investigate Aβ binding to wild type andvariant A2M (Narita, M., et al., J. Neurochem. 69:1904-1911 (1997); Du,Y., et al., J. Neurochem. 69:299-305 (1997)).

[0392] For the gel-filtration assay, Aβ1-42 is iodinated with ¹²⁵I,following the procedure of Narita et al. (Narita, M., et al., J.Neurochem. 69:1904-1911 (1997)). 125I-Aβ (5 mmol) then is incubatedseparately with treated and untreated A2M samples as well as treated anduntreated A2M samples that have been activated with methylamineaccording to the method described above. Activated A2M (Sigma) is alsoincubated with ¹²⁵I-Aβ as a positive control. A ten fold molar excess ofAβ is used and the samples are incubated in 25 mM Tris-HCl, 150 mM NaCl,pH 7.4 for two hours at 37° C. Controls containing only ¹²⁵I-Aβ are alsoincubated. The A2M/¹²⁵I-Aβ complex is then separated from unbound¹²⁵I-Aβ using a Superose 6 gel-filtration column (0.7×20 cm) under thecontrol of an FPLC (Pharmacia). 25 MM Tris-HCl, 150 mM NaCl, pH 7.4 areused to equilibrate the column and elute the samples. Using a flow rateof 0.05 ml/minute, 200 μL fractions are collected. Having standardizedthe column with molecular weight markers ranging from 1000 kD to 4 kD,A2M/25I-AP fractions are counted in a γ counter to determine the elutionprofile of ¹²⁵I-Aβ. If treated samples of A2M bind ¹²⁵-Aβ, ¹²⁵I-Aβ canbe detected by gamma counter at two peaks, one corresponding to themolecular weight of the A2M/¹²⁵I-Aβ complex (about 724 kD depending onthe polymorphism), and one corresponding to the molecular weight ofunbound ¹²⁵I-Aβ (4.5 kD).

[0393] In some embodiments of the present invention, immunoblotting maybe performed. For example, immunoblotting may be used to confirm theresults of the gel-filtration analysis. In immunoblot experiments,unlabeled Aβ with A2M samples as described above. After incubation, thesamples are electrophoresed on a 5% SDS-PAGE, under non-reducingconditions, and transferred to polyvinyl difluoride nitrocellulosemembrane (Immobilon-P). Two membranes having parallel samples are thenprobed with polyclonal anti-A2M IgG and monoclonal anti-Aβ IgG.Immunoreactive proteins are visualized using ECL and peroxidaseconjugated anti-rabbit IgG. Molecular mass markers are used to determineif the immunoreactive proteins from the anti-A2M and anti-Aβ blots forcorresponding lanes display the same mobility. If the immunoreactiveproteins display the same mobility then it will be concluded that Aβbinds the A2M sample.

Example 7 Screening Potential Therapeutics by Analyzing the Activationof Polymorphic A2M

[0394] To screen for therapeutic compounds capable of activatingpolymorphic A2M, unactivated tetrameric A2M from the media and extractsof the transfected cells is treated with 5, 20, 50 or 100 μg of testcompound in Tris/HCl or sodium phosphate buffer at 37° C. for 2 hours.Untreated unactivated A2M, and untreated A2M activated with methylamineor trypsin are used as controls. For example, A2M positive controls canbe activated by stirring A2M in a solution of 100 mM methylamine at roomtemperature in the dark for 30 minutes. The methylamine solution is thenexchanged for Tris buffer using a desalting column according to themanufacturer's instructions. After the incubation with the testcompounds, the activation of A2M can be determined by methods such asELISA assay or gel mobility shift analysis.

[0395] An analysis of A2M activation by ELISA is as follows. Microtiterplates are incubated for 2 hours at 37° C. with 50 μl of LRP (10μg)/well, and then rinsed with deionized water. The plates are thenfilled with blocking buffer and rinsed. 50 μl of treated A2M, untreatedunactivated A2M, or untreated A2M activated with methylamine or trypsinis added to each well and incubated for 2 hours at room ternperature.After rinsing, 50 μl anti-A2M IgG conjugated with MUP in blocking bufferis added to the wells and incubated for 2 hours at room temperature.After rinsing, MUP substrate is added to the wells, and incubated for 1hour at room temperature. The amount of A2M bound is quantitated with aspectrofluorometer with a 365 nm excitation filter and 450 nm emissionfilter.

[0396] Alternatively, the activation of A2M can be monitored using a gelshift assay. Activation of A2M increases its electrophoretic mobility ona native polyacrylamide gel. To determine electrophoretic mobility, theA2M samples that were incubated with test compounds and A2M activatedand unactivated controls are run on a native 3-8% polyacrylamide gel(Novex) at 75 V for a sufficient time to allow separation of activatedand unactivated forms. The gel is then stained with Colloidal Blue usingthat procedure recommended by Novex. Activation of A2M by test compoundscan be determined by comparing the electrophoretic mobility of activatedand unactivated controls with the electrophoretic mobility of A2Mincubated with test compounds.

Example 8 Screening Potential Therapeutics by Analyzing MultimerFormation of Polymorphic A2M

[0397] To screen for therapeutic compounds capable of modulating theability of polymorphic A2M to form multimers, A2M from the media andextracts of the transfected cells is treated with 5, 20, 50 or 100 μg oftest compound in Tris/HCl or sodium phosphate buffer at 37° C. for 2hours. Untreated A2M and wildtype A2M are used as a control.

[0398] To assess the ability of the test compound to modulate tetramerformation, treated and untreated A2M samples are run on a native 3-8%polyacrylamide gel (Novex) under nonreducing conditions, at 75 V for asufficient time to allow separation of the tetramer from othermultimeric forms. 10 μL of prestained molecular weight markers (BioRad)are also run. The proteins are then transferred from the gel to apolyvinyl difluoride nitrocellulose membrane (Immobilon-P) byelectroblotting at 100 V for 1 hour. The A2M samples are then detectedwith polyclonal A2M antibody (Sigma) using standard Western blottingtechniques known to those of ordinary skill in the art. An A2M sampletreated with a compound capable of inducing tetramer formation producesa band at 720 kD.

[0399] The ability of the test compound to modulate dimer formation canalso be determined using the above method except treated and untreatedA2M samples are run on a denaturing 3-8% polyacrylamide gel (Novex)under nonreducing conditions, at 75 V for a sufficient time to allowseparation of the dimer from monomers. An A2M sample treated with acompound capable of inducing dimer formation produces a band at 360 kD.Monmeric A2M produces a band at 180 kD. In the disclosure below, severaldiagnostic embodiments of the invention are described.

[0400] Although the invention has been described with reference toembodiments and examples, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

[0401] All references cited herein are hereby expressly incorporated byreference in their entireties. Where reference is made to a uniformresource locator (URL) or other such identifier or address, it isunderstood that such identifiers can change and particular informationon the internet can be added, removed, or supplemented, but equivalentinformation can be found by searching the internet. Reference theretoevidences the availability and public dissemination of such information.

1 15 1 88624 DNA Homo sapiens 1 tctttgcatc caatactcca acttctctgtggctgaccaa agaattggca cctatcttgc 60 cagtcaggta gttctgatgg gtccagcacagactggctgc ctgggggaga aagacagcat 120 tgatttgaag tggtgaacac tataactcccctagctcatc acaaaacaag cagacaagaa 180 ccacagcttc ctgcttctcc ctgagaagagaaaggattgt tagaatctcc cacaacctcc 240 aacaaggctg attgatagga accttctcctatacaagact agtctgtgaa gaatgggaga 300 ggtgccttcc tttgtctaat gcagaggcaacaacacagag agtcaaagaa aatgaagaat 360 taggcaaaga tattccttta aagaggaacaaaatacattc tagaaattaa cactaatgaa 420 atggaattat gtgatttact ttatggagaattcaaaataa ttctcataaa gatgctcact 480 gaagtcaaaa gaacaatgta tgagcagtgagaatttcaac aaaaccacaa aaagtatcaa 540 aaggtaccaa gcagaaatca ttgagctgaagaacacagta acttaaaaat tcactataag 600 agttcaatag caaactagat aaagcagaagaaaagatcag ttaatttgaa caccagtcat 660 tggaagtagt tcagtcagaa gagaaaaaaagacaaagaaa taaaaagtgt agaaaaccta 720 aggaacttat gtagcaccat caaattgaccattatacaaa ttatgagagt cagaaaagga 780 gaatagaaag agaaagaaag aaaaaacttattcatagaaa taatgactaa aaccttctca 840 acctgaaaaa ggaaatggaa tccaggttcaaaaataacta agtaagatga acccaatgaa 900 atccacataa aaatacataa tcattaaattatcaaaagta aaagagaatt ttcaaagcaa 960 taagagaaca gtgacttgta agatagacaagatgcctgat aagatgatca gctggttttt 1020 cagcagaaat ttgcagtcca gaaggcagtgaaattattca cagtggtaaa ataatacaaa 1080 cctgctaacc aagaatacta tacctgggaaacctgtccat caaaaatgga ggagtaataa 1140 agactttctc agacaaacga aagctgagggagttcatcac ctctagattt gtcttaccag 1200 aaatgctaaa gagagttttt caactgaaagaaaaggacac taaacagcaa cacaatatca 1260 agagaaggta tgaaactgat tggcaaaggcaaatataaag aaaaacacat gatactgtat 1320 tactgtaatg ctagtaagtc acttttacttccagttaaaa gttaaaagag aaaagtatta 1380 aaaataacaa actaaaatat gttttaaaacataaaataga tatcaatttt cacaaaaata 1440 aagtgtgtag ggacagatga taaagggcaaagtttttgta tgtgattaaa ctgaagttgt 1500 tatcagctta aaacagactg ctataactacaagatatttt gtgtagactc caaggtaacc 1560 ccaaaaagtc tataaaagtt acacaaaagacagagattta aaaatcaaag tatattggta 1620 cacaaaaaaa caataaaaca caaggaagacaggaagagag gaaaagacag acaaaataat 1680 tacaaggcta acataaaaca actaacatggcaaaaataaa tcttccccta tcaacaatta 1740 ctttaaatgc aaattgagta aacctcccaatgaaaacaca tatagtgact gaaaagttaa 1800 aaaaacagac ccaaatatat tctatatacaaggtacttac tttagattta agaacacaca 1860 taggctgaaa gtgaagggat gaaaaagatatcccatgcaa atggtaatca caagagagta 1920 ggtgacaggg caggagtatc atcatcttggacaagcactg gcattttaaa gttcccctta 1980 atcaaaaact gccccaaagg gcattggcctaatggctaac gtcagcatga ccataaacca 2040 caaatgacat ctctgaccag aaacattccaacacgaaaat aaaccctccc cgaccagaga 2100 tatgcctgcc ccaagataac ctcccctccggccagagaga tgtcagcccc aagataactt 2160 tccctctgac cagagacatt ccaaccccacaataaacttc tcctccacac agaaacattc 2220 caagcctgtg ataagctctc tcaccctaaaacccttaaat actcttagtc tgtaagagag 2280 agtggtcctg actaaaattg gccagaagcccctctcaggt ttattctcca aaataaacct 2340 gtctttgact gttgagccac taatcgtgtttctttcctct ttctttaact cttacatttg 2400 gtgccaaaac ccaggacggg tgttgtgggtagaggctctc ttgcaaccca ggaagcagtg 2460 ggcagtggca gctcatccca ctggatcctgagagtctctg gccaaccacc ccatcttgcc 2520 tcttacttca cttttcaagt gatttacatgagcaggacaa 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atctgttttattttgtaaca gaacatgatt tgaaaaactg 6000 gttattttac caaggctttg actggagaggtgtgctgtcc tttaaggaat caaacttgac 6060 ttatggagcc aataaaactc tcgggaaactggcctcatat tttatgtgca cagtccctgt 6120 acagggtttc tgacctgtgg taagtaaagaatgtcacttt ctgacaggcc agtaccccca 6180 agttatcttg gaacctcagg aggagaggaattcacccaac tcataggtat ttaatggtac 6240 aattccatga ctgggctcag ctttaaaaggccttatctca gattccttct atggaacaaa 6300 attccatcaa tgccagttta aaaggcctaggtaacaaata attattcttg ctgcactgta 6360 tgcaaataat taaaccaagt ataataatgcaaaccattcc taccatgatt tattttttaa 6420 taacggttac tggcagaaaa taacacgtggccctttccaa acatgtgcct ctgcctctca 6480 ttaggtaagg aatgttgctt ctatctcaaccaattgggcc gagtaagaaa cactgctaaa 6540 taacttaaag aaagggccaa agagctaagggaattccaaa acaaccaaat ggattcttga 6600 tttgggaaaa aaaccatagc atgggtcatcccattcctgg gcccccccct actactatgc 6660 ctaggactaa tgttcttacc ctgcctaattaatcttttcc agagattttt aactgacagg 6720 atcatggcca tttcacagac aactacccaaaaactgcccc aaagggcatc agcctaatgg 6780 ctaatgtcag catgaccata aaccacaaatgacatctctg accagaaaca ttccaacacg 6840 aaaataaacc cctccccagc cagagacatgcccatcccaa gataacctcc tctccggcca 6900 gagagatgca gccccaagat aacctcccctctgaccagag acattccaac cccacaataa 6960 acttctcctc cacacagaaa cattccaagcctgtgataag ctctcttacc ctaaaaccct 7020 taaatactct tagtctgtaa gagagagtggtcctgaccaa aaattggtca gaatcccctc 7080 tcaggtttat tctccaaaat aaacctgtctttcactgttg agccattttt catgtttctt 7140 tcctctttct ttaactctta caggaggagtggctacactt atatcagata aaatagactg 7200 agttaaaaac tgttacaaga gaccaaaagggaaattttat aatgataaaa gggtcaattc 7260 aacaggaata tataacaatt acaattatatatgcatccaa cagtagaaca cctaaatata 7320 taaagcaaac attggaaaaa cagaaaagagaaatagggag caataaaata atagtaggaa 7380 acttcaatac tatttgcaat aatggatacatcattcagac agaaatctgt atccataaga 7440 aaacagcaga cttgaataac acaatagaccaaatggacca aacagacata tgcagaatat 7500 tccaccaaac agcagctgaa tacatactcttctcatgtgc actcagatcc ttccccagga 7560 taagtcacat attaagtcac aaaataagttataacaaatt tagaaagatt gaaatcacac 7620 caagtgtctt tctggccaca acaaaattaaactataaatc aataacagaa ggaagactgg 7680 aaaaataaaa aatacataga tattaaacaacagactcttg aaaagtcatt gagtgaaaga 7740 aggaatcaag aaggaattta aagtgccttaatacaaatga aaacaaaaat aaaaaacact 7800 aaaattatgg tattcagcaa aagcactattaagaaggagg tttactgtga taaataccta 7860 cattaacaaa gaacaaagat ctaaaataatcaacccaaat ttatacttca agaggctaga 7920 aaaagaacaa actaagctca aaattagcagagaaagaaat aacaaaaatt agagaagaaa 7980 taaataaaac aagaaaagaa aaaaatcaaagaggctcagt gggttttttg aaaaaaataa 8040 ttaacacacc cttatttaga ctaaaaaaaaagaaatgaaa gaagatacat tataactgct 8100 cttttagaaa taaaaatgat cataagtgaacaaatatgtc aactaatcga ataacctaga 8160 agaattgtgt aaattcccaa atacacaaaacatgtcaaaa gtaaaaatca agaaagtttg 8220 aacagaccta tcattagtat ggagatttaatgaataacaa aaatccttct aacaaataaa 8280 aacccaggat cagatagctt cacaggtagattctaataca cattttttta aaaaagtgcc 8340 aatcagtctc aaatgcttcc aaaaagtgagagaacacttc caaactcatt ttataaggcc 8400 agtagcacac tgctaccaaa gccagacaaggacactacaa gaaaaaaaaa atgacaggcc 8460 aatatctctg atgaatatag atgcaaaaaatattttaaaa atattaggaa atagaatcca 8520 acagcacatt aaagggatca tacatcatgaccaagtagta tttattccca ggatgcaagg 8580 atggttcagt atgtataaat ttgtaccacattcacagaat aaagggaaaa aaaatcacat 8640 aattatataa atacaagcag agaaagcatttgacaaaatt caacattctt tcatgttaaa 8700 aactctcaag aaactataaa taggagagtatctcaacata atacaggcta tatatgaaag 8760 gcccatagat aatatcagac tcaacggtgaaaagttgaaa gcttttcctt taagaacagg 8820 agcaaggcaa tgatgcccac tcttgccacttttattcaat atagaaccaa agccctagcc 8880 agaacagtta ggtaagaaaa ataaattaaagcctaccaaa tcagaaagga agagataaac 8940 tttttcctgt ttgctgatgc cattatattatgtattaaaa atcccaaagg ctccatttta 9000 aaaaactgtt aaaactaata cacaaatacagtaagattgc aagctacaaa atcaacttac 9060 aaaaatcagt tgcatttcta tacactagcaataaactctg aaaaggaaat taagacaaca 9120 atcccattta caatagcaca aaaaagaattaaatacttaa ggaaaaactt ttccaaggaa 9180 gtgaaagacc tgtgtctgga aacaaaaacattgatgaaag aaattaagac acaattaaat 9240 aaaaatatat accatgttca ttgattggaagatttactat tgtcaaaata accataatat 9300 caaaagcaat ctatagattc aatgcaacccctgtcaaaat cacattggta ttgtttaaaa 9360 aaatagaaaa ggaaatccta aaatttatagggaatgagaa aacaccacaa ataacaaaat 9420 caatcttgag aaagaagaag aaagctggaggactcacact tcctaatttc aaaatttagt 9480 acaaaaccac agtaatcaaa acagtatggtgctggcataa agacagataa acatcaatga 9540 cagaatagag atctcaggaa taaatgcacccataaaaggt caactggtct ttgacaaggg 9600 taccaagaat acactagggg gaatggatagtcccttcaac aaatggtgtg gagaaaactg 9660 tatatccata agcaaaacaa taaaatttgatgtttatctt acaccataca caagaattaa 9720 ctcaaagtgg attaaagaca taaaagtaaggcctgaaact gtaaaactga tataagaaaa 9780 aataaaagac atgctttatg atcttggtcttggcaatgat ttcttggata tgacaccaaa 9840 atcacagaca acgaaaacaa aaacaaataagttgaactat gtcaaactgg aaagctcttg 9900 caaagcaaag gaataatcaa caaagtgaaaagacaacata tggaatggta gaaaatattt 9960 gcaaaccatg tgtctgacaa ggggttgctatccaaaacat ataagcaact cctacaactc 10020 aactcaacag caaaaaaact aataacatgactttaaaatg ggcaaggatc tgagtagaca 10080 tctttcaaaa gaaaacatac aaatggccaataggtatatg aaaaaatgct caatgccact 10140 aatcagggaa atgcaaatca aaaccacaatgagatatcgc ttcacacatg tcaggatgcc 10200 tattatcaaa taaaagacaa caagtggttggcaaagatgt ggagaaaact ggaatctttg 10260 tatactgttg gtgttaatgc aaaatggtgcaactgctatg gaaacatggt gtatatattc 10320 atctatattt gtatatatgt atatgcatattatatataca tgcatatata tatgcacaca 10380 tatatgcaca cacacatgca caatggaatattgccttttt taaatgccaa gataagaaac 10440 aatttattac agaagaaaaa tttctcatccaaaatataga aatcaataca actttgccac 10500 aatcaatata cacgaactgt acaaatgtatacccattcat aatttaccaa ataaaagatg 10560 attaacaaag ttcacaaaat agatgaaaatacttttaccc aggaaagtta caaaccagac 10620 ctccaatttc taaaatagaa gtttactcagtcttagaaaa ctacaagcta gcaaatgtac 10680 gtagagctgg ctggtgccaa caccacagttgaaacagtct ttctaagggt ctttttaaaa 10740 acccgttgcc atggcagatt ctggtcacttgctactttca aggtcaaaaa cacaatacaa 10800 agtctgacca ttttcccagg tcatgtttgctagcttgtct ttatgtacat ttagaaatat 10860 ttgctaggta aaagtcttgt cgtaaaatttccagtactac tatgtttaaa acgttgagct 10920 cccctattga gctgccaaaa aggtaaacaataattttcaa gtgtgatagt tcaaattcct 10980 ctgcgagatc tactacagag aaaggttctttgacatacgg attttcttta aaggaattga 11040 tgtaaaaatt taagtatgtc tgggagaagctgaaatcact ctaggacttc actccctagc 11100 aaataaagtg atcatttact tggactcataggctattaaa ttattgaaag atactgtaca 11160 aactatggca ctgtcacttt taaaaaaatgtttaccactc tatcttgtgc cggatcttca 11220 cagctgtgac atggtttaaa ttccataatccatccccaat aggagcccac ccaaagccaa 11280 aatcaaattt atccatgcac tataagatgatccatcttaa cctgatacag tcatcatact 11340 gtagtttttg gaagggctgg ttctgcccaagagaaattcg tccttacagt ttattcagct 11400 gtctaccatt tgtatgtcgg tgctgttttgagtgctaccc cctgctggtg gggctttcat 11460 acagcacaca gatggagcca tcttctccaattctgcagga cagacatctc ataggttgag 11520 gtgagcgtga gtccaaccca gagtgtgagttcacttggga aaagcttgaa cagctcctga 11580 ctgctcggtc caatccactg tgctgcctgtccaggggatc catttcatgg ttgatgcgaa 11640 tacaaagata acttgatctt ttgtatggcttttctgggaa tcagtgatgt ttataatgtt 11700 ctgtcagcag ttcctgcagg ctgtggctgaaggtctgcag ctgttgcccg ttcgtgagcc 11760 ctttgctgtg gagaaacttg gagacgaaggacatggaggc ggccatctcg cctaccctgg 11820 tggtggctct ggggtagaag ggatgcaaggaggcagaggt ggggcggccc gggaacgccg 11880 gggctcggct gcgctgcctc agcagctgagcagcccccag ggcttcctca cagagcagag 11940 ggctgacagg aagaaagggt gtgaggggcagacagctact tttttctttc attagagtaa 12000 aaaagttatt ttctagacag gaagaggcagaagagaggaa gagaggagcg atggcggttt 12060 ggttacatgg ctaggacctc cccagctgcctccacccctg gctgcagagc ctctgaagat 12120 cccaacagtt gcctttccag caccgaagggcgaacttctt taaaaagaag aaaattctgt 12180 atacgacaaa actaatgaat cttgaggacattatgctcag tgaaataagt cacagaaaga 12240 caaatactgc atgattccac ttacatgatttatctaaaac agccaaattc atagaatcaa 12300 agagtaaaac agtggttatc aggggctaaactgaagaggg aacggggagt ttgtaatcaa 12360 tggacataaa atttcagtga agcaaggtaaataagctctg gagatttgct ctacaatgtt 12420 gtacttatag tcgacaataa tatattgtacacttacaatt tgttaaaagg gagatctcat 12480 gttaaatgtt cttaccacaa taaaataaaatttttaaaag aaaaaagaca cctggtgtct 12540 ttttcttgta gcaaccgaag taaatagagatgtatgggtg tataggcatt agtttgattt 12600 tctcttacga agaggtctgg attatagcctccatcttgag accggagtca agcacttgag 12660 gtggggaatg atggagagat tggaagtggcattcttctcc acaacaccat gggttctgta 12720 gtcccttgtg caattctgca attctgccatgatagttatt gagaccaaga caggcaaagt 12780 ctaaccaggg aaagaagagt tctgtagacttccttccaat tctaccaagg ttgatattga 12840 gagcaggctt cttgccagca gcattaaacccaacaatcta tacttcctag gacaatctct 12900 acctttgatt tctgtaaaat aacacttttctgtttttctt ctgaggcccc tcctccacag 12960 accagtttct ggatatcttc ttctttatcagatctctaaa tgttgagatt cttctagatt 13020 tagatctatg ctgcttgttc tttgcactctacattttctt cattcatttc tacaactttt 13080 aatagtgtgt ataggtagct cccagattgctattttcacc gtgattctct tgcctcatac 13140 ttttcccctc tttggtgatg atgtgtcagcacattggccc tctctctaat cttgaatact 13200 ttatttctgt ctggggagtt tagcacatgcttccctctgc ctataatgct gtataacttg 13260 ccctctccct ttttgcaagg ataaattcccagcttaaatg ccatatcata agagaggtct 13320 tgactgaatg cacaatctat gttggatctccctggatgtt cagggaagaa aagaaacatt 13380 tttttctttc acagcattta tcatcatatggaattacctt atttaattta tttgtttata 13440 tactttgatc tgtattcttc cacaggaatgtataatcccc aatggcagga acatgtggtt 13500 tttgttaacc acagaacagt atctgatacaaaatatgcat taaaattctt gtgaactgac 13560 taaataatat ccatctctcc acttgattgctttacttgcc acacatcccc agtacctaat 13620 tcaagagctt ggctcattgt cagcctttaaatattgactg aacccaatta atcacttttt 13680 tctttaactt ctgaactctg tgcccagctccctacttgaa tagcttcact ggatatctca 13740 aaagaacctc aaattcaata tgtctactttccctatctgt tattagcaac aaacccagca 13800 cttgccctag taggaaatct acaatcatcctttacttatc tctaaaatga aaaaaaaacc 13860 cacacctttt tcataaagtt ttgagttcactaagtgaaat aagcaaggtg cctatcacaa 13920 tgcctggcat acagtatcta taaatattagtttagctata ccagttttca ttcattcaaa 13980 gaggcacatc ttttcacatc tctgaaatcaagatgtgttt tgtgatcagt agtgtttcat 14040 aattggatat acatctaatc attaatcaggtggaaatttt ttctttaaat actttattag 14100 aaaattgtgt cctttacctc aatggtgcttcagatgtgat aatttgcagt aacacaactt 14160 ctctctccaa tgtgcccata tatattaaagtagttattag gtcacatcac gtacatttga 14220 aatccatatt ttttctcact cttcccactgttgtaatctt actttgggcc ctcattttct 14280 tccaactgaa cactgaatta gccccctgaatggtcttcct gctgactata ttgcccttct 14340 ccaatgtgtg tgtcatattc atgccagagtaatcatgttt ctctcatgcc taactcactt 14400 cattatctac acaactgcct gcagaatggaaaccagattc tttcacatgg cacacagggc 14460 tctgactctc tctctcttta gcctaatccctaagccacag tcaagttgac ctcctttctg 14520 tacttgcaga tgacttaaca ctgtatgttgtttcacaacc catgtcgttt atcacactga 14580 cctatcttga aaatattttg tatcagctggaatccctctg gcaataaata acagaaaagc 14640 aactagcagc tgcttaaaca aataggaaagtccataggtt ggtgttccaa ggttgataga 14700 attgcccaat aatgctgatg aggaccaaggcccattttat ctttcagact tccttaacat 14760 gcagaccttc atccccctgc ttgttactttgctgtctcaa gatggtcatt gaaatattaa 14820 atatcacatc tattttccat taagtaagaacaaggaagga cattcagggg tatgcccact 14880 acaactcttc tttttaagaa agcaaatgtttcccataagc cctacccagc agttttctac 14940 ttatatctca ttgcccaaac agcaccatagtcactctggt ttgaagatgc ctagagagag 15000 ggatgttgtg ggtagtttag ccaatgagcagtgtctgcca cagtctgctt ggaaaatatc 15060 ttctcatttt ttgtgagatt caactcaagagttatatctt agaaatttag aaatgtgtat 15120 atatatgtat ataaactata tatgtatataaattatattg atatttatat aagcatacat 15180 aaaacacaaa tatataaacg tatatatagtatgtaccctt tcatcattaa ctgaatattt 15240 atcaaacacc tgtacacagt gcgtgctaggtactgttcta ggcactggga atacagcaat 15300 gaacaacatc ttatcctttg tggaatttacatcctagtgt ttaccctttg atctaacttt 15360 ttcacctcta gtaacttacc ttacagactatacctcaaaa acaaggatgc tcattgaagt 15420 gataactaga ataacaaaaa tctgattaaatatcactatg ggattgatta taatatacat 15480 aaaattataa aatgtaatta ttcaaattacctcttaatag agatatacta acatggaaag 15540 ctgtccccaa catatatttt aatttaaataatggaagacc aaaatgtata gtaaactctc 15600 acttttaaaa agaaaatcca tccccaaattcatacggaat ctcgaggaat ctgaaatagc 15660 caaaacaact ttggaagaag aataaaggtattagactcac acttcctgat ttcaaagcat 15720 atacaaagac acaatgatca aagcagtgtggcactggcat aaagacagac aaataaacca 15780 tcgtaataga ataaagagcc gagaaataagcctgtgtgta tatggtcaaa tgatcttcaa 15840 caagggtgcc aagaccactc attgagaaaatgatagtctc ctcaacaaac ggtgttggga 15900 acaatggaaa tgaaagttaa acctttattttacactatct acaaaattta acttataata 15960 tattaaagac ataagcataa gacctaaaactatgaaattt ctagaagaaa acagacagga 16020 taatctcatg acatggggtt tgtcaatgacttcttagata tgacaccaaa agcacaggta 16080 acaaaagcaa aaaaagataa atgggactacatcaaacttg aaaacatttg tgcatcaaag 16140 gacacaatca aaagagtgaa gggcaacatacaaaatggga aaaaaaaatt tacaaagaat 16200 atatttagaa gttattatcc acaatatataaagaactccc acaactaaac aatgaaaaaa 16260 aaatcaaata actagatttt aaagtgtgcaaaatatttga atagatattt ctctaaagaa 16320 tatatacaaa tggctaataa acattgaaaaggtgctcaac attactaatc accagagaaa 16380 tgcaaatcaa aatcacaatg acatactacttcacatctgt gaggatggct gctataaaaa 16440 aaaaacagaa agtaacaagt gttggcaaggatggggacaa attgaaaccc ttgtgcgcag 16500 ttggtgggat tgtaaaatgg tttaactgctatggaaaaca ccgtcaagtt tcctcaaaaa 16560 attcaaatag aactaccata tgatccaggaattttactta tgggtgtata tccaaaagaa 16620 ttgaaaacat ggtcttgaag agatatttatataccacagt catagcacta gtcccaataa 16680 ccaagaagta aaagcgagcc aaatgtccatcaatagaaga atgaatacat acaatcaaat 16740 attattcagc cttaaaaaga aatgaaattctgatacatgc tgcaacatgg atgaactttg 16800 aagaaatttt gctaagtaaa ataagctagtcacaaaatga caaatactgt attatttcac 16860 ctatatgaag tatccaagcc aattcataaaaacagaaaga agaacgctgg ttaccaggga 16920 cagagaagag gagaaagggg aagtgtttaataggttgttt agtagttata gagtttcaga 16980 tttgcaagat ataaaagctc ggaaaatctgtctcacaaca atgtgtatat actttacact 17040 actaaactgt acatttaaaa atggttcaccaggcgcggtg gctcacgcct gtaatcccag 17100 cattttggga ggccgacgcg ggtggatcacaaggtcagga gatctagacc atcctggcta 17160 acacggtgaa atcccgtctc tactaaaaatacaaaaaaag tagccgggcg tggtggcggg 17220 cgcctgtagt cccagctact cctgaacccgggaggcggag tttgcagtga gcagaacgcg 17280 ccactgcact ctagcctggg cgacagagtgaaactccgtc tcgggaaaaa aaaatgggta 17340 aaatgggaaa aaggaaaacc catacctgcatcttcatata tattattata tacaataata 17400 ctgaaaatac tttcagttta tgaatttggtgggtagctca cacattactt tatattttct 17460 tcatacttat atattttata ataagtatacattacttttt atagtcagag aaagattaga 17520 aaaaatttaa aaaattacaa ggtactttcttctctatctc tctcctcaaa tagtaatgac 17580 catttatttg tacttatgta gtcaccaacagaatttatat gtattgaatg aaataataaa 17640 tgtatcatca actagtgata atgaatgccttcaataaaaa tttatttata tttagcacca 17700 aattaagaaa ttaatttaat atagttcttcatttaatgaa taccgattgt tattaagtgc 17760 ctaggatatg cccgatgtgc ctgtgtaggttttgagtaat aaacatggtt atttcttgaa 17820 actatctttt gatgaagaaa gagaaaaagagaaagagaga aagaaagagg ttatggctag 17880 ctggtaacat tcaaaagcaa gccacttcatgaaaccccat tacaagtcaa gatagtatag 17940 tccaggtgtg tagtactctg gtggtctggttttatcatag ttagagccta aatacatagc 18000 aaagagaaca gatttcacat atttcaaaaacatgtaacag cgagtatgaa atacccgcag 18060 acccagtcac cccactccag cttctgccagagaaaactgg tgtaagaaaa actggtttgg 18120 aagttaattg cattcttccc cccacccccttgggccataa aattttatta gcaggtaagg 18180 ggaagagcca ggagtaaggg tccctcccttcccatcccct acccaggatc ctccctctga 18240 aggagagcag aagcccagga gcctgccactcagcggacct tgcaggtggg caggttgctg 18300 taaggatctt tctggcgctc gagcaggatgcggtcggcag tgatctcctg ctcaggcctc 18360 ctctggcagt cagcacacac attgcccagctggacttcgg caccgctgct cagagcctgg 18420 atccgcacca gctgggcctc aacgcaggcctccgttcctg cagtacggtc ttccaatgcg 18480 gttttcatgc tgagctgcga ctgcagctcaatctcaagac cccggagggt acgccaccgg 18540 tccgcgactc ggacttgtgc atctggagctgctccgtgtg gccagcgacc tcccagctca 18600 gatcctcagt ctggctggtg accaggcttcagcatcttgg gcatctgctc agttatgacc 18660 gcatactggc ttcacgtgtt gcccgggatcttggcaagac caatgcctgg agcagaatcc 18720 acctccacac tgacctggcc acccgcagcactgatttcct cgttgtggtt cttctccagg 18780 tgatccagct cctcctgcag gccttggatgtgcgtcttca ggtcagccct ggccggggtc 18840 agctcatcgg gcgccctgcg caggccttgatgtctgcctc caagctcctt cgcagagact 18900 gctccatctc agacttggtt cggaagtgatctgcagccag gcggcgggtg tcaatctgca 18960 agacaatccg ggagttctca atggtgacaccaagaatctt gtcccgcagg tcttcaatgg 19020 tcttgaagta gtggctgtaa ttgccggggccccggggccc agtcgcagat cttcaccttc 19080 agctcacccg gcctcctcca gagcccgcaccttgtccagg taggaaccaa gtggtcgtgg 19140 atgttctgcg tggtgatctt ggtgcccgtcagcagcctgt cggacctgca gaggacgctg 19200 gcatcgccgc tgccatagcc ccagaggaggatgagggcac aaagtgggtg gaggacacag 19260 acaggctacg gcttcggagc cccagtggatgctgggctct aggaaggcgc ccccgactcc 19320 aaagtgcacg gagccgctgc ccagcccccaaaggactaag tggccaatga ctggcgaaag 19380 ctgcaggaag ttagggtgag gcagagaacggttcaaggag ctgcaatcgg caggaggaat 19440 cggaaatttt aattgcatta ttaccaattataagaatttt tgtgctatga gtgggttttg 19500 tgctatgagg ggacccaaaa aagggctgataaaacaccaa aaccactggt gaggagaact 19560 taagagggaa ccaggcttca agctcctaaactcaccatgg actgccacag aatttaaagc 19620 acagtccata ttcctcagaa agctaaccaagcccaccact gggtgtccta ccaaaggccg 19680 ttccctggca ttattctaga cagaacaacccagacggcaa aaatgtactg ctgtggagag 19740 atgaatcaat tcctaggagg actggacagacattctcaaa agtttgttgg aaagatagca 19800 gagaagagaa gaatgaggtg ctttaataaagggataggaa gaaggtgaga acaaggaact 19860 gagattagga gctgagtgtg tccatgacagaccgcctaga aagaatgtag tgtctagagc 19920 ttttactcat agaccgcggg acaaggtatatttaccatat agctctgtat tttccctctt 19980 gtgttataat gttttctgta attaattactctaacccttt tagcaaagtc taatacactt 20040 cagcattgtc tcgtgatgat atacagactaaatggacagg tgagtaaaag aaaaaattaa 20100 ggcgtcctca ctggtgggag cagaatcccaggcagagaac tatgcacaga agtttcaggc 20160 agtgatcagc tgaaagtcag attgcagtccattgtattcc atggtggaga caagaacagg 20220 gagggagcac ccatacttaa aagaaacttctaggcttgga gcagtatggt attttataca 20280 catcacctct gttagtcaag ctttggatgggggctagata gcagcccact gaaagacctg 20340 gagtccctgt attcttaatt gctatgtcggaaagttccat atttagaaat cagatgggca 20400 gcaggtaagt taggcattct tctgaaaaaaattaagcctc taccaggact ctcgcttggt 20460 ttataaaatg agccacagaa gaaacagcaaactcaagttt tcctctcaaa aagtcctgtg 20520 tcacttgaaa agttgcttca tacacattgtgtcctaaata tgaaatctaa agttgttact 20580 gttttgaaat aaagtttcaa gagaatatattttatataac attcaaatga gttatagtaa 20640 aaatgctagg tcttaagaga ttcaccggatgaaataacat tctccacttc agaaaatgaa 20700 actagtacaa tattaaatac tgtgattaataataatctct aatatttcta catacttttg 20760 ctcctaacag ttgagttata tgctcaccctgtatgattgt ggtatattct gttatgccag 20820 ttgcattatt gtaattataa atttgaggaataaagtacac gaacttataa aatataaaca 20880 tggccaggca cagtggctca cgcctgtaatcccagcactt tgggaggccg aggtgggtgg 20940 atcacaagct caggagtttg agaccagcctgcccaatatg gtgaaacccc gtctctacta 21000 aaaatacaaa aattagccag gcatggtggtgagcacctgt tgtcccagct acttgggagg 21060 ctgaggcagg agaatcattt gaacccgggaggtggaggtt gcagtgagcc aagattgtgc 21120 cactgcactc cagcctgggc gttagagcaagattccatct caaaaaacaa acaaacaaac 21180 aaacaaaaaa tatacataca cgcactattttaaaactcag tttttatcta aaacctagat 21240 taaaagtctt tggaaagagt ccatggagaggaatacgtta aaaatgccat tgaagccatt 21300 ctgcaatgta taatatttca gaatgacatgttgcacacaa tgtctacata atttttcgtc 21360 aattgaaaat aatgtgatca attggattaaaaatatatca ttaactcatg aatttaaata 21420 tatttaatga gcttaagttg attgtattttctgtatttct taaactcaag gtattaccta 21480 gtgctttgag gtgtcgattt tctggccagaaacttctgcg gctggcgaca agctcttgtc 21540 ctgcatccag gaagaatgac aggaagaatgaggtacacga acaagtgaag ggtgcacaat 21600 aagaatgagg tacccagaca agtgaaggacaagatgaaga tgagctttac taaattttag 21660 aacagctcag aggagaccca cagtgggtagctcctctccg taggcaggtc ttcccaacat 21720 ctactgctct cagcagagag gaggccctggagcgggtgct ccttgctcct ctctgcactg 21780 tagtcccgaa gtctctgcag gtctctgaagctctcagcag agagggtagt tcctctgtgc 21840 agctggttgt cccatcgtct cctgctattagcagagaggg cagcttctct ctgcaactgg 21900 tcttcctgtc cctccatcct ctctcttgctctgcctgagc ccgaggcttt tatggggtgg 21960 aggaaatgcc tgctgattga tccatgggcagccataggca ggccaaagga ggcaccacaa 22020 gtcctaggga ctgactgccg ggatcccaaccttcaggtcc tccctggcct gaaggtgggg 22080 ccttacaggg gacctgcccc cttctgcccaggagtctgcc tgcctcccgc tgccattcat 22140 ggcccctggg ctcagcccca agattggagcaggctctggg agaggagaaa ggccaggcaa 22200 tgggagcaga cacccctgag cctgcagggtcgagagaagg ggagggtctt cccggctctc 22260 gagggtgcag gctgcagaga tgcctggacctgcacctgtg agggtgccgc agctgcacca 22320 gggaatctcc cgcgcagcca actgggaatgggcaggtctc ccgcttgtcc cgggctctct 22380 ctggctcagt ggagcagtag gcccaggtctgcagccactg gtcggggtgc tgcagccgca 22440 cggggagtgt agatcttgcc tgctcccagccccttccaag agcacaggga ggctcagatc 22500 cacagccgca gtttgggcgg ggctcctacctgctccatag agcaggaggc cgcggtctgc 22560 agcctcggtt tgggcagctg cagcggcaccggggagctct ggccccaact cagaagggac 22620 ggagctccca ccggctccac tgcagccaacattatggcag cagcggctac catcaatggt 22680 atcccatctt tggcaagtgg aaacatcttaatgaatttcc cgtttgcccc gagaatcctc 22740 ttgtctctga tcctaaggta acatcacatacatgtctgtt acactaggat tagagacaag 22800 ttctgtttag aaataactcc aagaacagcttttatatttt attttcacat tgaaaatcag 22860 tcagatttgc tccagcctca aagaatgtgtttactaaaat taaatgaatg ctggcaggga 22920 gctgcacttt ttttttctaa ataggaaatgggttaagggc ggcagctgag tcctttcgac 22980 ataaccctaa tagtctttgg cagcatcctttctgtttggt aggataagat attctaggct 23040 catcttgcat tttccgcgaa aaaaaaaaagccattgaatt aggcatagag gaaattacta 23100 taaagtttaa gggataatgt aaaggatctaggaaaatact ctaattgctt cacaaacaag 23160 gaagaaactg aaactagaaa tgtagtcaattagtattgct gatgtgcttt atgcaagaaa 23220 gatagtatat ttgttctctc ttatctgcagcttcactttc tgtggtttca gttacccttg 23280 gtcatccagg gtccaaaaat attaaatggaaaattccaga aataaacgat tcataaattt 23340 tcaattgtgt gccattctga gtggtgtgataaaatcttga cacccgggat atgaatcatt 23400 cctttctcca gcgtatccat gctgtatatgctgctgttat tccccctaac ccccacccca 23460 gtcacttagt agctatctca atactgcagggcttatgttc aaataatcct tattttactt 23520 aataatggtc ccaaagctca agagtagtgatgttggcata ttgttataat tgttcttcta 23580 ttatcagttt tgttaatctc ttactaagcctaatttataa attaaacttc atcctaagta 23640 tgtatgtata ggaaaaaaca tagtatatatagggttaggt actatctgtg gtttcaggtg 23700 tccactgggg atcttggaac atatgcccctcggataaggg gaactactat atacaagatt 23760 aaataaggtc acttaaaatt catttataaaattatgtcct aaaatatttg taccagtaac 23820 aagatatatt caaattattt tatctaataaaaattaacat gttttatttt ctaataaaag 23880 ttaaaatatg ttagcagtct ataccacttctttaattaca tggaaaaaat tacattatga 23940 tgtctaatta ataaatagtt atttataattactgccaaag tcaaaaacca gtttgatact 24000 tacatcttaa ataccatgcc tttttatacccttttcttta taaataactt gtaatttaac 24060 cttcaaatta tactgcatta ctttaaattaaataatgaga gtacgtatgt ataacttgct 24120 ttgaaaataa atgaattatg aaagctacactatttaaaac ttgagaactt tcaataatac 24180 cttatacaat atattccata attactttttaataaatttt tataatatta ctgtaaattg 24240 tgggtttagg gaccgacagt tcaactattagcaaaataat cttttagcat aacaaaaatt 24300 ggaacatcta aatctcttag aacttgtcttagcccattta tatagtaagt actacgtaat 24360 tgctagctgt cattacctgt tactgttgtagctattgttg ttagcctcat tatcatcagc 24420 atcatctgag taattaagta aagttggcaaacaactttat ctttcctact tttaaaattt 24480 aaccccagtg tttgctccca aactaagtagaactctctaa aatgaaagtt ctgacactgg 24540 aaagttccac agcgaacatg agcactacagtgaacaaagg ggctcctatg gttaagggtc 24600 atgaaccaac cttgtgcaaa cccacacacaaacacgcata cacttatttt ctgttcatca 24660 aaggaaaagt aatcctgtaa catttcccccaccttgcttc ctattccaaa ataaactgca 24720 gatgaagatt aaaatctaaa cacaaatacttaatgtagga aaaaatgcag gataatattt 24780 ttacaatttc agtatgcata tgcagaaggatttcttaatc agaaaataaa gatacagtat 24840 aaacaaaaag aaagttaaac aaagataaattataagcaac agggttccta caagtgacaa 24900 tgatgctgaa catcaccagt aatcatgaacttgaaaaaga aaagacaaaa tagatcatgc 24960 tcccctcccc atcagattgg taaaattttaaatgagtgct gggaaaatat atacacataa 25020 gcattgctag tgagaaggta agttggcattttagaaagtt gatgagatag tatctataaa 25080 tttgaaatgt atatacatag caaattcacaatggggacag attcattata caaaaatact 25140 tcggcatgcc ccaaagtata aatatgttaggacagccatt gaattctatt gtttgtagta 25200 aattgtttta gtccaaacac taattcctctgtagcaaaca taggatctaa taaaatggat 25260 tatgtgtgga aatcagtcct ctttagaaacctaaaggacc aagtgtatcc tgattaaaaa 25320 gataaaacgc tttctttctt tctttttgtttttgtttttt tgtttgtttg tttcgagaca 25380 gaggctcgct ctgttgccag gctggagtgcagtggcgtga tctcggctca ctgcaacctc 25440 tgcctcccgg gtttaagcga ttctcgtgcatcagtctccc gtgcagctgg gactacaggc 25500 gcacgccacc acacccagct aatttttgtagtttaagtag agacggggtt tcaccatgtt 25560 ggccaggatg gtctcaatct cttgacctcatgatccacct gcctcagtct cccaaagtgc 25620 tttttgataa ttttgagaaa tgatggaggcatattagaat gaaaacaacc tgaggatgtg 25680 cttttatctt tgtatattca aatattttttctcattaaaa agcagaaagt ccgggtatga 25740 tggttcatgc ctgtaaccct aacactttgcggggccgaga taggaagatc ccgtgaggcc 25800 aggactttga ggctagcctg agcaacatggtaggaccctg tctccataaa aagcttaaga 25860 aaaaaattag cggggcgtgg tggagtgcacctgtagtctt agctatttgg gaggctgaga 25920 tgggaggatc agttgagcct aggagttcaaggctgcactg agctatgatc taaccactgt 25980 actccagcct gggcaacaga gcaagaccctgtctctgaaa aaaaaaaata cacacacaca 26040 cacacacaca cacacacaca cgttagtgggatagcacaaa tgagaaaaac tctgctcttt 26100 gatcactgag tacatctctg tagatatatatttccttcac tgcagatttt gcccaagata 26160 cttcgtcaaa gacaaagcca gtacaccctctaatagggtg aatatggtta tgccacctac 26220 tgagcttgtt tttgatacta gttaatatgtaaccagatga aattgtcatt atcgtcactg 26280 tcaggactat gggaagctta agtgttctcttttcaaggac aatgtgcgct aactgtacaa 26340 ttggtacaat taaataagtt atattcagttcctgggaagc actatagcaa tacaaggaga 26400 aaatttgatt ctatttattt ttgttaaggcccacctacct cctaatccta atttctctca 26460 tttcccaaat attccttgtt tgttcttactgttatgtgtt ttcctgtatt ttgctcttct 26520 actttctttt ccatggacta tctttttcccttcctttttt tcgctctacc cctttacctc 26580 agctttctag cagtatttgc taaatacttcaaaactgtat agaactggtt caaattgtgt 26640 gctccctttt ctgtcaagaa cttgctactcaggtaaccca attggtgatt tttcctggaa 26700 acactgatgg atgctgttcc tatagcgaaacccagaacag agatgaaata gatgtcatcc 26760 tcagccatta gcattcaaac tataaaaattaatttacact ggtatagtaa ggatcagaat 26820 gtcaaagctg tgttacacct agcatcttgtatgaaactac cccattaagg tgagaccaca 26880 gatattattg ccccactatt ggcatgaaagctgaggctca gagcagttaa ctgagttacc 26940 caggaccaca cagctaagtt agaagtagggctcaggtgtc ctggcaacta actggtccag 27000 ttattttttc tctcaagctc gttttccctctcctaaagaa taggaggctc tgtcgtggtg 27060 aaaggcgatt ttagtaatac tttcctttttatctgtgatt ataatgaatg cggcatctct 27120 cccattaagg atcattcctc cacccacattcttaatacat ctgctgcatg catccttcag 27180 agacctccct ctgggatcat cccttctcactccaaaaagc tcaacttctc ccctgtcatt 27240 tgtacctccc actcagcatt tttagaagcaatatttcatt caaacttatt caagtttatt 27300 tccacctaaa gaaatattcc tttcaccctggcatctccgt caggtactgc tctgttgttt 27360 ttctcccctt cagacaaact gccaaactggctctagttcc tcacattccc catcaccctc 27420 agcaagcttc tgccccacac cggcactgaaacagctgaat cccaatgtcc ttgtccttaa 27480 acccagcaga aaaaaaaaat caatcaattatttgatttca cagcggcact tgacatgggt 27540 agccaggaat ttatcaatga caacctttacagatcatctt tgtaatttat catgaggcat 27600 caaatgaatg ctattaacat taatccctcctattttaagt cattaatcca agtaaatgct 27660 cacttatttc tagcgtctta gaaaccatttaaattatgtt acattatgaa tcaatacatt 27720 ataaaattat accatcattt gtaataattttttaaaatgt tgtgtgctat taacattgat 27780 gccttggtat aaagtcatga tcattctggtctagtagcaa tcttctattg actattctct 27840 tactaaagcg gtcccttccg tgggactcagagacctcaca ctctcctgcc tgtgtttctt 27900 cctctctaat tggcccttct tgctccacttgggtgctcct gcccattgcc tagacaagag 27960 cattccctgt aactctgtct tgggctctttttctcttttc atcaacatct tctacgtggg 28020 tattatcatc catttccatg gcatcagcttgcccaataaa ctgataaatc catagtctct 28080 ataagtacag cagatctcat caagctagtggcattcagac tgctttaact ttaaccaaaa 28140 ataagggatt ttgtacatgt tcaataagcagttcccactg tgacactgta atcacatttt 28200 cacaattgtg acctaggaca cttagagtaaaggatacaga tgattgagac agaaatagtg 28260 acaaagaaaa ataaggttag gatatagattttaatgctgt aacagacctc aaaatacaat 28320 ggcttaacta agagaatgca tttctctgtcacataaaggt cccaactggc gtagactttt 28380 gatgactcaa gggctcaggc tgtgcctggtttgtggttct gccttcctta acacatggct 28440 tccatctgat gagctacagc agtacctatcactagtcagc atgtccacat tccagcctgg 28500 gcaaggaaga aaggggaagc gcagaactgtacccttcctt ttttaagtca tgaactgaaa 28560 gttgcatgta tcacttccac ttgccctccagtcaccagaa cttagtcata tgccataccc 28620 agcttcaagg gagtgggtta aaaacatagaagtcaactag gcagtctgca cccagcaaag 28680 gatcgggagt tctattatta aagcagaattggagaagtgg taacaggaaa caaccaccag 28740 cctctgctgc atgtatatga aacagatgtttcccaaatca ctattctcac ttattctgtc 28800 tgatacactg tattttttat tatattctctttcatttttt aaaatcctgg tcatgactca 28860 cagggcatga tgttacaacc cacttagatgctaacaccat aatctgaaaa atattaccta 28920 tattatgtct aatattggcc acttgaagtatggctagcct aaattgatct atgttgtaag 28980 tataaaattc acaccagctt gtgaaaacaaattatgaaaa aaaagtcttt aagatatcat 29040 taacaatttt atattggcta aatgttgaaatgatcatatt ttggatatat tggattaaat 29100 aaaatacact attaaaatta atttaatgtttctctttatg tggttactag aaaatttaaa 29160 atttaaaatt acacagggcg atcacattctatttctagta gaccacactg ctgtaagctc 29220 aagattcaaa tgtcaaactc ctgtgaatattaatacgtga atatcccaca agcacttact 29280 ccatcttccc aaccctcagc ccttctgtcctccttctgct cccaccaatc tgtgtttctt 29340 ctgtttcact cacccagcta aaggcaacacaattcactcc gtgacgagcc aggaaaatgg 29400 aaagacacat tttcctttat tcctcacattgatatattca ctgagcacta taattacctc 29460 ttaaatatga tataaatctg caagctcttttcaataccac cacaaattcc atagttcaaa 29520 atgccatcag ctttcaccta tattattacaccagctccca tctggtcttc ctgcatcctg 29580 gatcacctct ttctagctgc cctttcaaatttcaataaga gcaagctttc caggaaacaa 29640 acctgaagtc aatccactga gtactcctctgaatacctta atattgttga caaattcctt 29700 tctgatttga agtatcagaa aggaatatttcctccatacc aaatagtttt catttcatgc 29760 atgtgccgtg attcttctcc ctcctttgcatctgtcattc gttatgctta gaaagctctt 29820 ttcatctctt tgttcttcga gacaaccactactcatactt cagagcttaa tttacatttt 29880 gctttccctc aaaatttttt taaaaggttccaggtctggg ttatgtgctc tcttatgtgc 29940 tcccagagca tcctgaactt ctgcaataatatgtttggct actgtatttt atacagtagt 30000 tttatattgt attttatact gtattttatacagtaggtgt tatattgtat tttatacagt 30060 agttgttttt ctgtctgttt ttgccccaacaagaatgtaa aatctttaag tgcctgtttt 30120 catacttatt tgaccaccct atctctagaatcttgcatga tgtctagccc tagtaggatc 30180 aaaaaatact tacaaagcaa ctgaatagctacatgaatag atggatgaat aaatgcatgg 30240 gtggatggat ggattaatga aatcatttatatgacttaaa gtttgcagag gagtatcata 30300 tttggaaggc agtaaggaag tctgtgtagtcgatggtaaa ggcaattggg aagtttgtta 30360 ggcacaatag gtcaaaattt gtttttgaagtcctgttact tcacgtttct ttgtttcact 30420 ttcttaaaac aggaaactct tttctatgatcattcttcca gggcctggct cttcatctgc 30480 aacccagtaa tatccctaat gtcaaaaagctactggttta attcgtgcca ttttcaaaga 30540 ggactactga attctgatgt ggcttcaaacatttaggtta ggcatatcta atggagaact 30600 tgcagccaca ctgacttgta gtgaaatatctattttgagc ctgcccagtg ttgcttaaat 30660 tgtagttttc cttgccagct attcatacaagagatgtgag aagcaccata aaaggcgttg 30720 tgaggagttg tgggggagtg agggagagaagaggttgaaa agcttattag ctgctgtacg 30780 gtaaaagtga gctcttacgg gaatgggaatgtagttttag ccctccaggg attctattta 30840 gcccgccagg aattaacctt gactataaataggccatcaa tgacctttcc agagaatgtt 30900 cagagacctc aactttgttt agagatcttgtgtgggtgga acttcctgtt tgcacacaga 30960 gcagcataaa gcccagttgc tttgggaagtgtttgggacc agatggattg tagggagtag 31020 ggtacaatac agtctgttct cctccagctccttctttctg caacatgggg aagaacaaac 31080 tccttcatcc aagtctggtt cttctcctcttggtcctcct gcccacagac gcctcagtct 31140 ctggaaaacc gtgagttcca cacagagagcgtgaagcatg aacctagagt ccttcattta 31200 ttgcagattt ttctttatat cattcctttttctttcctat gatactgtca tcttcttatc 31260 tctaagattc cttccagatt ttacaaatctagtttactca ttacttgctt acttttaatc 31320 attcttcccc aactctctga agctctaatatgcaaagcct tcctaagggg tgtcagaaat 31380 ttttagcttt ttaaaagaat aaattttagatattcacatt catattgatc tacttgagac 31440 catgctattt atcttttctt atttcctctttctcaagggt ccattttcta ttttataaaa 31500 ataaagacaa ttctctccca caaccaaacatggaacaatg ccctggagta taaaaatcta 31560 tagagtgcca aataaaggaa caatttgaaatactggtgtt gatattgaaa aagcaaggga 31620 ctctaatgtc agaagagaaa tccttttgcagatgaggtgg tgatgaattc tttgtttcaa 31680 cacaactgaa ggaggaactg aaggaaataccagctgatga gtgatgagaa gggattcttg 31740 ataatagagt actaggtgat ttttggcatgtaatgcagaa gttgcaagaa gtggtaacaa 31800 tgatgcaatt gttttacctg ccatttatttacttttatgt gagccattct tcttagcact 31860 tatagctaca caaaacaaaa atagtaacagaattaatgtt gtttaattct tgcaatccat 31920 ggatgcataa attcactggg ggaaaaaacagctcatcatt ctcattaaag atgtgcttca 31980 aaagtatttt aattttatat ctaatatgtatgaatcatac tttgtattta ttttgttttg 32040 atcagttata tacaagtatt tttgaacatagctcagtcag aaggaaatgt ttaatattta 32100 taaatttatg gttacattct atttaaaagaggagttaaag ttaaatttac ctacccacat 32160 atgttacata tatatgtatt tatgtatatgtattcatata tgtatatatg tgaacataag 32220 tatacatacg tatatgtata gatgcttgacaataaagaag taagaataat tcacaacatt 32280 ttttgaaata taaaaattta ggataaatttctgtatggta attggcatgg aaattcaaat 32340 tcaaaaagga aaaaagaaga gaaagatattaaatatcaga ccattaaaag aattttttaa 32400 tgtactttta aatagtgata gtaggtatcttatactacag tgtttattat tcatgagaaa 32460 attgtaaaag taatctaagt attaatttaaaatatcatca aaaataatat cttttgctat 32520 tacttaaaat catgataaaa atatgtttacttgaaaatat gtaaggagtg cacagagtcc 32580 aaaaattatt ttaggagttc tgtgagcaaaaatgtataaa aactacaggg ttgatcttaa 32640 attacatgtc agggtactga gaaagtttctgtactgcaca tgagttacca aggtctaaag 32700 tcaatcacca gaggaccatt tttggatggagccattgtct aatcatgagc tgaaaggcaa 32760 atatttaaaa tgcaaacatc catggttaggtaacactctt aagaccttat tagctgctta 32820 ccacaactga gactgtgaag taatggctcactttctttga ggctcagatt ccatatctgt 32880 ggagtggaag cgagtgctta ccatacaattttcacagggc tgctgaatgt gtgtatgtat 32940 gtatgtgtgt atatatattt taatgaaaattctataattt gattagtttt tgtaatgtcc 33000 gcatgactga gagcttgctt actttttacagcaacttgaa ggtaaaaata gattttacaa 33060 catgaacaaa tgtaactaca tatttttatttgaattcaga tgttcacaaa ttgttcctta 33120 aagtgaagca tgcctacaag ttttaatctgtttaagacct acctcaagta aaatgttcac 33180 tgccatggca tgtgagggaa aagggaaataattcttatgc atggccttca acggccaaat 33240 ttcatgctca tcagtacatc ttctcttggtgtagaactga tgatgataat tatgatgatg 33300 gaaaaaagtg ctgttgatag caatgcctctcttccttcac tttcctctaa ctgaaccgtc 33360 tcattcccag gcagtatatg gttctggtcccctccctgct ccacactgag accactgaga 33420 agggctgtgt ccttctgagc tacctgaatgagacagtgac tgtaagtgct tccttggagt 33480 ctgtcagggg aaacaggagc ctcttcactgacctggaggc ggagaatgac gtactccact 33540 gtgtcgcctt cgctgtgagt gtggctgtttgacttaatat acttggttct tttagtcagg 33600 gtcataggga tctagtattc tgtcagatgaggctttggga ggttggtaag aactgcagga 33660 aggaatccaa atgtagcaaa ctaagtatagaaattaagga gcaatgcatg actctccagc 33720 catgggagac aaatatttac aggcaaactaaaagtcagct taataatcac atagaaaccc 33780 tattagccag aaaggaaaaa aaaaaaaagaaaatgtgact tcttaaaatt aagatggaaa 33840 aaatattaat aaagcaaact agaccaaggctcaacatgag ccacccgtgg tggactgaca 33900 ggaacacatc actccacact acttctaagagtaaaaattg tagaaattac cctgagcaaa 33960 ggtgtaatat ctcttcagag ttgtccacatgtgagatgct tacagcctag tgtcagtaaa 34020 atacggggtt tttttccata gcctgtaaaacatgcttcga ccatgccctt ttagcatgta 34080 atatcagcct ggtaaagctc agcgtataattgaatcaaga ctgtttctgt gagctgtatg 34140 aaggtgtgaa tctcacttac aacctctcttactgatttat tttcctccac cttgtgtcct 34200 gtctccccat ttgtaaaatg gcagaagtgattcctgtcca tgccaacttt ccctgctgag 34260 aacaagggag tgaagttaag taagaagggcaagatctcca gggaagagtg caacagcaaa 34320 taggggagga gacctgcggg tgctgaatggggtttcagga tcggtcctgt atttcaggtc 34380 ccaaagtctt catccaatga ggaggtaatgttcctcactg tccaagtgaa aggaccaacc 34440 caagaattta agaagcggac cacagtgatggttaagaacg aggacagtct ggtctttgtc 34500 cagacagaca aatcaatcta caaaccagggcagacaggta tgaagaagcc tacagacagg 34560 acaacttcaa aaaggaaaga tcttcttcccctggatgttc cccaggcaaa gttcctataa 34620 tcttggttcc ttaatagctt gtcttaccagcctacaggcc tactttgggt ttgggggctc 34680 atgaaaaata tttctgtttc agtgaaatttcgtgttgtct ccatggatga aaactttcac 34740 cccctgaatg agttggtgag ttttctattatctacataaa atgattgtct gtataaacag 34800 gctgggaacc tgttttttgt gctgagggaagaccagggag aggaagaatc tggtatcatt 34860 aacagtaact tctggcatta caacagcacaagatcctaat ctaaaacatc attccaggta 34920 aagaaagtag gtaattcttt ctgtcttggtgctggtactc agtcagttgt cacacaatta 34980 aatttacttt tcggatggtt cttaattaggacaattagaa agatacattc aatagcagac 35040 acagaaaaat cctcaaagaa cctaagctcaaaaaacattt taaagattta gattttttct 35100 atacacatct accaaaatct tcactataaaggaaagtcag ggtaattaat ttgttcctca 35160 agactaactc ttggtatctg tgataagaaacagttctttc tattgtaatg cagacatcaa 35220 cccaaagtct tcatttttct ctcccaaattaacttcttca cattttctta tctcaaaaag 35280 aggcaactct tctcttctag ctccaaagacaaaagattat ggcctagttc ttgtttctct 35340 ttctctcata cccacatcca cttcactggaaaatcatgtt ggcttaaaat atattcagac 35400 tatttcttat catctgaact actgctgcaagctagtccta gtcaatgtca tctctaaata 35460 agatcattac aataaccttc aaagtggtctcccagcttct actttcactc ctctgtctaa 35520 aatgaggcac cacacccatc actctgtcagcctaacttgg ctttgttttt ctatttgcac 35580 ttaccaccat tctatacgta ttgatttttaaaaatctgcc tatttctatt ataacataag 35640 ctccattaaa acatggttta ttgttcttttgtgcgtggtt atattctcaa tacctagaat 35700 gatacacagc gcatgagaag gtacttaataaagattagtt tttaaaaatg aataaacatt 35760 catagtagct tcttatcaat gtttattcatttttaaaagt aatatttatt aaactaaatt 35820 tattaatgaa atatgtccat tcctcctcattcttagaact acgtaagaat tctatgaccc 35880 atcttaatga ttacatctga acatatttacttatagttac ataagtgttt atttctcaac 35940 ctgtaaaatt gcatagcaaa ggattgatcttcactaggat gctataagca cttaagacat 36000 gttaatccat ttttagtaaa tggcactttacatgtatatt tgttgctgaa ggcctagtaa 36060 gttcttaaca ttatttatta attgcttaaaatagattaat gaaaagttct ataaaattta 36120 atctggaata tttctgtatt tcactatagagggaattatt ctatatgaaa ctaatttaga 36180 ttttttaaac tttttattgt ttttaatttttgtggaaaca tagcaggtat atatatttat 36240 gggttacatg agatattttg atacaggcatgcagtgcatg ataatgatta tggatgataa 36300 tcattatcat tatccatatc attatccaatcatggataat gattattatc atgcattgca 36360 tgcctgtatc aataaatgga gtacccatcccctcaaacat ttattctttg tgttacgaac 36420 aaatccaatt atacttttag ttatttttaaatgtacaatt aaattatttt ttactatact 36480 caccctgtta tgctagcaaa tactagctgctttgcttata aatgagattt aagaatattt 36540 gaaaataatt ataaacttct tttttcttttgtctttcaga ttccactagt atacattcag 36600 gtaagcaaca tgaaacattc catattaaaaggaaagcaat acatataggg aaaatgttct 36660 tatttcagag gtttttacaa tatctcagaaacttgtcatt aaaggagaag ccttcaaact 36720 cccatagagc tagatggcta taactcatctcctctactca ccctttacta ctaccccatt 36780 tgaccttttt gtagataact tagggtttccatagatatct tttattagct ccaatgctcc 36840 aggtgctttt gtaagttata attaatttactcatatagga tcccaaagga aatcgcatcg 36900 cacaatggca gagtttccag ttagagggtggcctcaagca attttctttt cccctctcat 36960 cagagccctt ccagggctcc tacaaggtggtggtacagaa gaaatcaggt ggaaggacag 37020 agcacccttt caccgtggag gaatttggtatggatcatga aaagtcatca agcattattt 37080 ttcttcatat ttaaactctt aggtcctggaatttaagttc atttggagtc tttccatttc 37140 ccatgggtga cattgggctt ggagtagaattaattacacc taagtccaat gaggacatca 37200 gtgatctgtg aataggactt cacatagcttcgttattttc tgtagcaata tttaatacca 37260 acccccaaaa ttaaaacatt cttgttttaatggagttttc cataattaat taagcacaca 37320 gtgatctctc atagtccctc aactgaaatcttcatttgag aggaggatag ataagaatag 37380 attggagagc agagctacct tttcagagccctaaaatatt attagggaac tgttacaggg 37440 aacctgaaaa taggaattcc cccaaagttgaaaaccaatc accaaccttc tttatcacca 37500 atcaacagtt cttcccaagt ttgaagtacaagtaacagtg ccaaagataa tcaccatctt 37560 ggaagaagag atgaatgtat cagtgtgtggcctgtgagtt cattttttaa aaatcttttg 37620 tgggggatta tttaaaagag actcaccatttgggatattt taactactct ctccgggagc 37680 agtggcaaca caaaaatttt aagtgctttgacagcatcct catctgtaga atgttattct 37740 cctgttgctt ttctattttt attttctttcacgttcttat cagtattatt ctatcatgag 37800 gtaagaaact gtttctagag aggttatgctaataggattg attctgaaag tgacaaaact 37860 gcacacacac acacacacaa aatgggaagggtggatagtg ttgaagggta ttggttctgc 37920 cttaaccaaa aataaccaaa cgtatattagggagataatt aacacatggc tataggggaa 37980 attcaatcat caacagatca tttacctgaccttgcatgct tactggaaaa atcacttaga 38040 ttcagaattt gtagagatag gcatacaactgaaatctcat ctaactcact agtccaccta 38100 aggcgggtct acatacctgt ctgcactgacatttttacga agagactgaa cgtgatttct 38160 caagacagtc tgttccaacc ttgaaaagtcttgtttcctg gatcttatgt tcccatccat 38220 ggtggcacac agtagcagta gcaggaagagcaggtatagt cctgtccaaa gatcacacat 38280 gtaatacacc tactattcag ctaggcccctgctccagttc tgtctcacag tgatggccaa 38340 gttcctggtt cctcataact ggtgtgatcttgcaaataga catacagagg acattctcaa 38400 ataggaaagg agacaaatgc acttggaaacccagcaattg tctatgacat atttacgaca 38460 caatcccact tttgtaaaaa agtaaacaggcccggcgtgg tggctcacgc ctgtaatccc 38520 agcactttgg gaggctgagg tgggcagatcacgaggtcag gagatcaaga ccatcctggc 38580 taacatggtg aaaccccgtc tctactaaaaaatacaaaaa attagccagg catggtggcc 38640 agcgcctgta gtcccagcta cttgggaagctgaggcagga gaatggcatg aacccaggag 38700 gtggaggttg cagtgagcca agatcgcaccactgcactcc agcctgggcg acagagcgag 38760 actccatctc aaaaataaaa taaaataaaataaaataaaa taaaataaaa taaacaagtg 38820 aaactcgata tagagatggc tgcacatggggagaggatga tggaagaaga gacactatcg 38880 tactaatagt ttgtattcct aggtagacttgagggcgttt gggttttttt tgttctgttt 38940 tgttttgttt gtttcgtctt gctggtattgtttctctttt aagaacgggt ttaagtttta 39000 taataagaaa gactttttta agataaaactaaaaaaaaac gaggaaaaaa aagaaatgat 39060 aaaagaagaa tgtaaatttc agcatgttgcaatggagatt tctataagag ccattagtga 39120 ctcttgtctt caatattgtg tgagcccagcagagagcaga gggaagtaca gacagggaat 39180 atactgtagc taaaggggag atataaatagctagaaaatg gggaggttca gtgtctgctc 39240 tgattccttt gggaatgttc tcatgacagatacacatatg ggaagcctgt ccctggacat 39300 gtgactgtga gcatttgcag aaagtatagtgacgcttccg actgccacgg tgaagattca 39360 caggctttct gtgagaaatt cagtggacaggtaggttgaa cactattttt tctagagaat 39420 agcgataaag gcattgttga aaagcagtgagttgcagcat ttttctgacg caggaagaga 39480 acaatctaga agagaattcc atgttggctattgtaatttt tcaaaaaaaa tcatgaactt 39540 agcacaatgg gaattattta tttctcgtaattgcccattg tgagtgtttc agaacgatag 39600 acactgagcc atctaaagcc tccatgggcattcacttcta caaaggaagg aaaaaaccat 39660 acacctctta attgccttag ctggccaggccatcagttct gctctctctg tgaacaagaa 39720 cctatcacat ggccccacca agatgccagagagttgacaa acacagtccc catatagaag 39780 gccgcttccc agccacagct gaatattatggaggaggaac cagactttga tgaggagttc 39840 taatggtcaa gagcagatgt actgtgtatttcaaaatagc aagtggacag gacttgaact 39900 atttccaaca catagaaatg atacatacttgagttggtag gcaccctaaa tcccgcgatg 39960 tgatcattac acattctctg catgtaacaaaatatctcgt gtaccccata aatatgtata 40020 aatattatgc atcctttgta caaaaaaaattactcctcaa attttaagac atttctatcg 40080 caatatatct agggaatttc agataccaaggaatacatct gtcaattata catgagatat 40140 tgttgtgaaa tttaatattt agttgctagagaatatttat tgtgtcctcg tcatagaaaa 40200 tgcctacatg atgttgtccc cccacaaaaatcacaccggt tatctgacca ctgatctatt 40260 tagtataata tgcattatat ttttgtattttattttctcc cttcttagct aaacagccat 40320 ggctgcttct atcagcaagt aaaaaccaaggtcttccagc tgaagaggaa ggagtatgaa 40380 atgaaacttc acactgaggc ccagatccaagaagaaggaa caggtttgtg tactacatgg 40440 gtataagaga aaacacaaca ggcattgattttcttagcca aataatgaat tgtaagttgg 40500 ggggaggtga tagaatttta gagacatcattcttcccaaa aataacagat ttttttctct 40560 tttttcagtg gtggaattga ctggaaggcagtccagtgaa atcacaagaa ccataaccaa 40620 actctcattt gtgaaagtgg actcacactttcgacaggga attcccttct ttgggcaggt 40680 ggagtatttt ccagttcact catcaacccatgtactgtta cctaattagc acaatagtta 40740 tggtttgtgc taaaaccatg cctggttaatgttatcattt aatataacca aaagtataaa 40800 atatcaccaa ggcttgatta gtataaccaaaggtataaaa ctacataaaa atagatttat 40860 tcttctgtaa atttgtgtat gaaatgtatgtaattatcct aaggccttat taaaattagt 40920 agagttttcc ccccttcttt tgaaacagcattgtacaagt cactcaatct ccttctcatg 40980 gtttaccagg cagtctcagg tttttatgacattttctcac aagaatctca aaattcatgc 41040 tgaccagttt cattacgatg ctaacactaactttgtatgg aaacaggtgg gtaggtggtt 41100 ttaattttta ttttgaagta ttaaagattctacaataatg tttatttcat ggatagtata 41160 tttacactat ttttctataa caagtatatttctaaaacag tgaacatggg gtaaaacact 41220 gctatttaag gttctcagat ttttgaattatgaattttca tgttatctac caaaaaaatc 41280 ttctcttaca atttttctgt tgtcagcagatgtgcaaatg gatctttatg actctagaag 41340 tctgagatca cagttacatg tgcctcaatgtgtatttact gtgtatcatt ttcttaatgt 41400 aaatgttaca ggatttcagc tatgaagggtgtaaaagagg catagactca acaaagtgga 41460 gtacattcta gaaggcttgg ttatgtcatgacaccaaaat gtattcaact tcctaaaatg 41520 aaagtgtagc tcatttgtaa atttctcagaaagatagtgt actttgtaag tacattttat 41580 tgctgttaaa tatcctattt gtcataagactctctagcta gaagaaaatc agaattatgt 41640 gcctattctt gtctttgtac atgcagccaacatttcactg gcaaacttag attttgtaaa 41700 tcaattatga gtactgatgg gggtcagcctattgttcttc ctgctaccaa ctgtagctta 41760 tactgaaaaa gaattgccac ctttacaatctgaactggac tactcaactg ccaatacaat 41820 attagcagta aattagattt ggcaacttatttaatactgt gtgcctcttt tcaattttat 41880 atttttcaca tgggaagtgt gtcataaaatctgcctacct tccaaaaggc tgaagatcta 41940 ggttgagaag cagagaccat gtctaaggcaactggagaaa cacacaggac aatgtattgg 42000 caattgttta cttgtgcact tatgagacttcagacactaa tctataggaa gttaatggtc 42060 cactccaaaa taggtgtttg gggcacaaataaatttagtt aatagattaa tagattaaaa 42120 tatttcatta tcatattact ctagtgctctgtgactctct aagagttata tataataata 42180 cacagcactt ccaaaatatc tagaagacatttttcaggtc actcttgtta ttatccctat 42240 cactctctat cccttcacac tgctctgtttttcttcttag gattgattac tactaaatta 42300 gtgtatgtta tgtatatatt tatttagcatctatctcttt cactagacag taagctctgt 42360 gatggaagaa acttcgtttt ttttcactgctgtgtcctca gtgcctggaa ccatgttcaa 42420 catagaggag gcactaaaaa atgtgataaatgaatatgtt tggtgcctat tagttattac 42480 caatataact aatccacagc ttttaatcttcaggtgcgcc tagtagatgg gaaaggcgtc 42540 cctataccaa ataaagtcat attcatcagaggaaatgaag caaactatta ctccaatgct 42600 accacggatg agcatggcct tgtacagttctctatcaaca ccaccaatgt tatgggtacc 42660 tctcttactg ttagggtaag tttggaaagaaattaccaat gacatgaagt agccttggaa 42720 acaaggttgc aacctaaggg tgagaaaatttccaaactgt gtctagttct atggagagaa 42780 aaaaactagc aattagaaac cgattgaaggttaacttttt taaagtttat gaaaagaagg 42840 cagtatattg tgattaaaag tgcgggttagactttagcag tgttgctggg aacgtaaaat 42900 ggcggagcca ctatgaaaaa cagtatagtagttcctgaaa aaattaaaaa atagaattac 42960 caaatgatcc agtaatccta cttctggacatatattcaaa agaatcgaaa acggggtctc 43020 aaagagctat ttgcacaccc gtgttcatagccgcactatt cacaatagct gagagatcga 43080 ggctacccaa atgtccatca agggatgaacaggtaaacaa aatgtggtat ataaatacaa 43140 cagaatatta tgcagcctga gaagggaagaaaatcctgtc acatgctaca gcatgaatga 43200 tccttgagga cgttatggta agtgaaataagctagtcaca aaaagaccaa tactgtatga 43260 ttcacgtaca tggggtttct aaagtagtcaaagtcataga aacagaaaac aggatggtgg 43320 ttgccaaggg ctagggaaag agagaaatggggaattcctc ttattggtat tcaggtttag 43380 ttttacaaga tgaaaagttc tggagatctgttgcacaaca gatatactta atactaccaa 43440 actgtacact aaagaataat gaagatggtaattttatgtt gtgtgtgtgt gtgtgtttat 43500 aataactttt ttgaaaagtg tgaattccagaatctgaatc agataaagtg gttaaaaata 43560 ctggctcaac cctattttaa ggaattaactaaaacctctg tgcttcattt ccttatctgt 43620 aaaatgattg caatactaac acctgactcttggggtggtt gtgaagatta agtgaaatca 43680 tacatgttga atcacttagt aagccccctttaactgttag atacttttac tataaaagcc 43740 aattctaaca taattagcat ttagttttaaatatatatac tccaaaaatt attaccttac 43800 tttattttgt cttgctattc taattttctccagatgacct gttcatacca atataagtta 43860 ttcggtatag catgcagtcc tctattcatcaagagagatg tagacatgtc tatagataca 43920 tggatataca atacatattt aatatatattacataatcat ggtaatggaa aacgcctgcc 43980 tctttagagt tttaccttgt tatagagaaataaataatac attttattta tttagcttta 44040 atagatggca taaaagcacc tccctctaatttgaatgttt actcatttca aaaagtgtct 44100 tgaatgcttt ccacgtgcca gcactgtgctagtcttgaca atgaaattat tatttctacc 44160 tgttccctgt ttggggtgat catatatgtgttgtcaccaa aatatacatt aagatatgaa 44220 caaaatgttt tgctcaagga ttcactagtgctattgggag ctggggatga aggtagagga 44280 agcctgaaag gagctaagga ataacttctacaggaagaaa agttggaaat ataacccaaa 44340 tagggcctct gaggtattag aatccagattgcagggagca ttaaaaatag tcagcctagg 44400 ccaggcacgg tagctcacgc ctgtaatcccagcactttgg gaggctgagg tgggtggatt 44460 gcgtgaggcc aggagatcga gacgagcctaggcaacgtgg tgaaaccctg tctctacaaa 44520 atatacaaaa aattagctgg gtggggtggcatgcacctgt agtcctagca actcaggagg 44580 ctgactggga ggatcacttg agccccagaggcagagattg cactgagcca agattgtgcc 44640 actgtactcc agcatgggcg ccagagcaaaaccctgtctc aaaaaaataa taataatagc 44700 aatcagccta agatagccca gaagaggtagacttcagcta attcatcagc tcagctctta 44760 aaccaatgct ttctaccaat gtcttctcagacccctaagg ttacaatatt ttatttattc 44820 ataataccca ttcaaaatcc accctaaaagaggcagctgc tttctgaaag cacagttctt 44880 ccatttctag agattattta ctctcctcaatgaagtttca tagctccagt gtctctaatt 44940 gcacaggtaa agcagtcaaa gaaatttcaagcaagctaat cagagcaaag gatgcctcct 45000 tttatgctct taagaaatat aaatctcaatcccaggaggc tctgcagtgt aaagtcacaa 45060 agcatgccta catttgaagc agagaaacaaaatcaggggt ccttctccca cttttcattg 45120 tggaacaaaa gatttctagc tactagttaaggtaggacag taaacttacg tagttttgtg 45180 agaacattaa tctttatgac gtataatctaaaaataatat aatttttcta attcattagg 45240 tcaattacaa ggatcgtagt ccctgttacggctaccagtg ggtgtcagaa gaacacgaag 45300 aggcacatca cactgcttat cttgtgttctccccaagcaa gagctttgtc caccttgagc 45360 ccatgtctca tgaactaccc tgtggccatactcagacagt ccaggcacat tatattctga 45420 atggaggcac cctgctgggg ctgaagaagctctccttcta ttatctggtg agaagggagg 45480 ttactgcgtt gacttcactg tagacaaaagctctctgtgg agcaagtaat catgaagctc 45540 tttagatgtc attacttcaa cttcttatccatgttccttt tgaaagtttg atttctcttg 45600 aggtgaatta ttgccggcag ggactcaataaaacaagtat attgaagtga gactgaagcg 45660 tgttctctct ggcacattta atttctttttatttcctttt ttgcagataa tggcaaaggg 45720 aggcattgtc cgaactggga ctcatggactgcttgtgaag caggaagaca gtgagtattt 45780 ccatcatctc tgcattgctg ccccattctgacccattcag ccttacacca cggaagtatc 45840 agaatacttt cccttttttt ccataggtttttgggggaac aggtggtgtt tggtcacatg 45900 aatgaataaa ttctttggta gtgatttccgagattttggt gcacccatca cccgagcagt 45960 atacattgta cccaatttgc agtcttttatccctcacccc cttccaccct tccccctgaa 46020 tccacaaagt ccattgtatc attcttatgcctttgcatcc tcataactta gctcccactt 46080 cctaatagct tagctgccac ttcctaatagcttagctcct acttctgagt gagaagatat 46140 gatgtttggt tttccattcc tgagctgggaatatcaggaa tactttcaag taacgagaga 46200 ccatttccat ctaatattaa ttaaaagaaagttgagggag ggaaaacaat aatggttttt 46260 ttctcaccct ttatcaaata tgtgttgtcttttttttcag cctattttct ttgcgttatt 46320 taaaatattg gtgtgggcca gctatgatggcttacgcctg taatcccagt actttgggag 46380 aacgggatgg gaggatctct tgaggtcaggagtttgagac cagcctggtc aacatagtga 46440 gaccccatct ctataaaagt aaataaataaataaataaat aaataagaac aaagggggaa 46500 aaataaaata cataaaatat tgttgtgtataatctatgct tattccagat ttttaaacca 46560 taaatattct tgaacctctt ctcaaaatgagaattgtgtt agagaatggg gtggagaaaa 46620 tgtacgattt caaggtatgt tgtatataccaggtcttgtg ctgagtcatt tatgtcattt 46680 atattattat aaatatcttt tttccctttcaagatagtta tgatttatga agcattaaat 46740 tgggtacttc taccgactga agcggactcttgccatgagc aaatatgtca taatataaac 46800 ctttgaaggt tgctcctagt ctagtagactctaaaagata acttgctaga aaagtatcag 46860 taatctactc tctcattagt tcttgtagcaacacagaagc accatagcag actgaaggaa 46920 acttaatgca acctctaaag aatctacataggcatcaaaa aataacttct gaaggagcat 46980 ggtgttcctc ttatggagaa agaaccaaatattggctcat agaaggttaa agagcatgta 47040 gcatgaatac aatgtgttga actcttactcttacatctca taacttcagg ttctatttcc 47100 ctcttctagt atgaggccta gatcaaatggtagtgaattg gatgtcttca tacccttaac 47160 tagcatcaga acattaggtg ggtgagctgaagcttaaagc tggtgaaaaa tcaacagagt 47220 atatgattta tgagaggaga gagggctttgatagggtttt tttctaaagc tagagtcctt 47280 agaggtaatc atccatttgt tacatctactgtttattatg tctgttacat ctactaatgg 47340 aaatagtcat taacctatga tggcttcctctcattctctt tgtgccccag tgaagggcca 47400 tttttccatc tcaatccctg tgaagtcagacattgctcct gtcgctcggt tgctcatcta 47460 tgctgtttta cctaccgggg acgtgattggggattctgca aaatatgatg ttgaaaattg 47520 tctggccaac aaggtgtgtg ttttagatcataaaatcttc aacatgtaaa actagaagtt 47580 actattgtta tcatttgttt tacacatgtgaacattaggg ccaaaagggt taaacaaatt 47640 ccccagagac attcagcaag gtagtggcagagtcacacta agaagcagaa tcacttgatt 47700 ccttatacaa aactctcaaa cttctcctagcagtgcctct taccaattac acagttcaga 47760 gtatgttatt cccttcttct atgatgaaacgttgggaaat atgtaagcaa gtttttaaga 47820 ctactaagga gccaaaagaa aaatgcaagagcacctagac ccatgcatgt cccaccacaa 47880 ttaatgagca ccttgccgtg tatcaaagataaaaatggca tttcatgact agtaatttat 47940 aagaataatt agaaataatt ctactggctcaagtgattct tgagaatgaa agagaagtgt 48000 agggaacaaa aagcttcagg attgacacaatgccaaccct ccacgaagtc aagcagagtg 48060 gttatacatt gtacatgagg aacactctgcaaatgcgagt gaatgcatgg gtaaacctgg 48120 aaatttgctt ttctgacctt ttgttccaatttcacaggtg gatttgagct tcagcccatc 48180 acaaagtctc ccagcctcac acgcccacctgcgagtcaca gcggctcctc agtccgtctg 48240 cgccctccgt gctgtggacc aaagcgtgctgctcatgaag cctgatgctg agctctcggc 48300 gtcctcggtg agttcctggc agcctcaggaatcaagaagg gccgtgccag gggctcagag 48360 caaggaaaaa tgactggata agtaggataagatcaattaa aattatattt atctaggaat 48420 taattgcttt gagctctctt gtgggctttcattggtaagg aattatatat atatatatat 48480 ataactactc acttctgcag ttaaaaaaataaacaaaata caatatattg aatcaaataa 48540 aagaattttt aaaaggacaa aatgtgatgaaaattaacaa ccaaaaaagg actcgttatt 48600 cagacacgtt aagcttcttg ctcaagagcacatagcaata caaattcaaa tcttctgatt 48660 gtgtcaggac atccgtatat gcaaggctggagagaataca gagcagattg tgaaaagtgc 48720 tatatgagaa gtgcagttat gcaaaataaaagaaaggatt aactgagcaa ccggggaagc 48780 cgttcgaaaa ttatatattt aaaatgtaaaaagaccatac agtacccaaa gtccttaaaa 48840 tcccagagct ccatgcaacc agtaataggagttgtcaaac tagtttcaac atttcaaaaa 48900 gcctaacaaa agtgatacat atacctgcactgaggctata caggctaaat gaaatatcag 48960 atttccgttt ttataagaat tatggctgggcctggtggct cacgcctgta atcccaacac 49020 tgggaggcca aggcaggcag ataacttgagatcaggagtt tgagaccagt ctggccaata 49080 taatgaaacc ccgtatctac taaaaatacaaaaattagtc agacgtggtg gcgggcgcct 49140 gtcatcccag ctactcagga ggctgaggcaggagaatcac ttgaacccag gaggcagagg 49200 ttgagtgagc cgagatcgtg ccactgcactccagcctggg caatagagca agacttggtg 49260 tcaaaaaaaa aaaaaaaaaa agaattacatcagttgaaat aattgctgtc ccaagctaca 49320 cataatttct gagggaacat atgtattatctctgaggaac aaaacttagg gaaattgaac 49380 tactatataa ttgacattga taactgaactctttgaaaat atggagctaa tagaagaaat 49440 accaaaagga tatgctgaat tggaacaacaaaaactagtt aatagcatgt actaatagta 49500 gtttgctcac atcttcaaat ttttaatatgagaccaattg taataacctc ttactgaagc 49560 tttttctact accataattt aacaaagagaaatttatatt tttattttcc tgaaatagaa 49620 acttataaaa aaatgtttct tacttgtttgtccttctaat gggattttaa ctgaaaatat 49680 tagaatactt ctaaaggaag catcaagtacatttcaacag taggatctca agaggatatg 49740 tgggaaaata atatacatgt tttatacttttataatataa ttttataatt ctcaaagaat 49800 ttttcaaatt ataacaaccc tgatgggtagataaggcaga cagtcctatt tattaataat 49860 aaagcagaag ctttcaggga tttagtagtagacctgagtt aaaacaccaa ctcttctcaa 49920 gctttgatta gtgtcttacc aatgaaacactttgctgcta ctagtgtagg tcattcattc 49980 aacacattta tttaatgccc actgtgttctaggtattata ctaagtgcta gtagagatca 50040 agcagtgagc tactggaaag ataaaaatgtatgtctcatg gaacttacat tgtctgtccc 50100 atagatgaga cagacaataa ttatgcaatatgccacaata aaagcaggga gaggaaatga 50160 gaaatgttaa gatactttga gaaagtgtctaatttcatca caccactcac tttgctcatc 50220 tgttcttgtc aatcagtttt aaactcctacgaatataatg caatgtaact atcacaattt 50280 ttatgtctgt cttacttctc accttcaaatggacttgaaa gcatcatgcc tagaatttta 50340 cggttaaagt tgtatgtatt atatgaagatctggagcatt ttgtttccac taataatacc 50400 taagaaaatg ccatcgtgtc ctgtggagagaggatattcc tattcgtgtg cctgtttaga 50460 acatgcaccc attaactttg ctatatactgagtcagttgc tcaccacaag ataagcacaa 50520 aactatcatt tccttctatc atctcaaagctttgtgcaat gtcacaaata cagcagacct 50580 cgatttttca attaataaag ttttatttcattccagtgtt gagtctagtg gtggcctctg 50640 aactgtgtaa cgaagtagta cttagtacttagatgagtac ttagatggag tgtttggttt 50700 ttcctaaaat tgttaaacat cttcaaaatgaaaacactgt gtcaagaaaa tgatccatac 50760 cctctataaa tcatcaaagc aatgagagcgctcaaagaaa gacggatgtt cattattcct 50820 gttcttttct ccttgaactt aaaaaatgtcacaaaggccg ggcgcggtgg ctcacgcctg 50880 taatcccagc actttgggag gccgaggcgggtggatcatg aggtcaggag atcgagacca 50940 tcctggctaa caaggtgaaa ccccgtctctactaaaaata caaaaaatta gccgggcgcg 51000 gtggcgggcg cctgtagtcc cagctactcgggaggctgag gcaggagaat ggcgtgaacc 51060 cgggaagcgg agcttgcagt gagccgagattgcgccactg cagtccgcgg tccggcctgg 51120 gcgacagagc gagactccgt ctcaaaaaaaaaaaaaaaaa aaaaaaaaaa agtcacaaat 51180 aagtttgcct ttttgtcttt cgtatttgtacaggtttaca acctgctacc agaaaaggac 51240 ctcactggct tccctgggcc tttgaatgaccaggacaatg aagactgcat caatcgtcat 51300 aatgtctata ttaatggaat cacatatactccagtatcaa gtacaaatga aaaggatatg 51360 tacagcttcc tagaggtaaa ctccttatgttgcagatggt ctgatcttaa gcttcttaaa 51420 atattacaca tggaaaagag tctgtatttgaatgccttca tgtcctagtt gagggtaatg 51480 ggatataaag aggtaagtgg cttctctactaatagcagca gatctgcgaa aagctgctga 51540 actaagctgg aactttttgg agtattattcaagttttctt ttttcacagg aattaattgc 51600 tctgtgatgt ttattaaaat cacatatacaaaataattac tgttgaaaga gtttaatgaa 51660 aagaataaaa ttacatcctt aagtatataaatatcccttc tacagatgtg aatgttaaaa 51720 gtttaattta agttaactgg attgcttgtcaaattcaata aaaagaagta gctacctatg 51780 tatattttat aatatatatt tactgattagatgataattt tctttgcagg acatgggctt 51840 aaaggcattc accaactcaa agattcgtaaacccaaaatg tgtccacagc ttcaacagta 51900 tgaaatgcat ggacctgaag gtctacgtgtaggtttttat ggtaaacaaa aaattaataa 51960 atatatattg cctaatatat tcaccaaattttaaattttt taaaagatac aatgtgacaa 52020 aaattaacaa acaaaaagga catgtgagtcatacatatta aggttattgc tcaaggtcat 52080 atagtaatat aaactcaaat tctagtaaatggaggtacat gtgttaggct gaaaggaaag 52140 agaaagtttc caagcgtagg attagtgtaaacagaaatag aaatgttcac acaacaacac 52200 tacattctcc atcagtcagg taaaaaagctgttcaacttc cccaaaacat cagccaatat 52260 ttatgttgga accacacaca ttcatcaatgacatgagcta cttctttgat gaactgaata 52320 gtaatcaagc atttatttat agtcccatattgtcaaatca tgattgagga taagttgagt 52380 acagagaaaa gagggtcaaa aatcaaggaaagcatttaag gagaatcagt tggctctatg 52440 gaattcacta tgaagcctac cgcatattttattatttata aattatattg tataatcctt 52500 tatcagtaag atatatagta aatttatgcatgtataggta tatatatata tatatatata 52560 tatatatata tatgcacaat ttttttttccagagagctgg ttgtgaaata ttgaccagca 52620 cacccttaat agaaggtgaa tagagtgaaggaaactaact gtaatcttct gacacgatag 52680 agaataaaaa gtctttatta ttattaaacttttccctcct gtaaactgtt tctcaaagcc 52740 actgtcataa catgtgtgtc agtttttccttgctcctgca gaggagcaca ccaatggaaa 52800 cttggattcc tgcccctctt cacggccttgtgtcataaca ctctccactt agcacagcgg 52860 cagcagccac ataggactcc acagagtccttgtgccccac agacaatcca agcctctgtc 52920 tgccaaagtc tctatagcct ctgcttatgaccctcctccc ccagctcact cctagcttat 52980 tcttgttttt ttttgttgtt tgtttgtttgtttgtttgtt tgagacgaag tctcactctg 53040 tcgctcaggt cgcccaggct ggagtgcagtggcgccgtct cggcctcggc ctctcaaagt 53100 gctgggatta caggcatgag ccaccgcgcccggcctctcc tggcttgtta agtcaaccaa 53160 catatttcct cctcaaagaa gctttccttgatgacccaag cgccctctcc tccttcctct 53220 ctatttcatc agacgatttg ttttttgttttttttttttg gtggcaacta ttacattctc 53280 tcataagctt tatctgtatg tttattgtaatgtcttcttc ctcactcacc atagagtcag 53340 atgtaatggg aagaggccat gcacgcctggtgcatgttga agagcctcac acggagaccg 53400 tacgaaagta cttccctgag acatggatctgggatttggt ggtggtaaag taagtaactt 53460 cctgcatatg caatatgcaa caatagaggtccctgactat tttcaactct ttgctagttt 53520 ttctgttttt attgttttat atgtttatgatgtacaacat gatgttttga tatatatata 53580 tacacataca catagtgaaa tgattactacagtcaagcaa attaacacat ccattagctc 53640 acattgttac ctatttttgt gtgcatagcaagcacaccta aaatctatcc tcttagccaa 53700 gtttcagtat ccaatacagt attatcaactgcagtcctca tgctgtactt tagatttcta 53760 aatttattca tcctacgtaa cttcaactttgtaccctttg acctgcttct tcccatccct 53820 gcaaccccaa ccacctttct actctctgttcctatgtatt caacttcttt tagcatccac 53880 atacaagtaa gatcacgcag tacttgtcctgtgtctggct tatttcactt agcataatct 53940 cccccaggtt catccatgtt gtcacaagtggcaggatctt cttccactta aggctgaata 54000 atattccatt acgcatagcc acaatttatttatccattca cccagacact taggttgttt 54060 ttatatcttg gcaactgtga ataatgctgtaatgaacacc aaaacacata tatctctaca 54120 aggtgcttat ttcatttcct ttgggagtatacccagaaga gggatttctg ggacatatgg 54180 tagtttccat tttaaaattt ttgaggtatctccatactgt tttccataat ggctgtggca 54240 atgtacattt gcaccaaggg gagtgaacaaattccttcca gagagacact ggaaacagag 54300 ttttatccgt gaaacaagcc agtggggcaggtggagagaa agagccggaa tggccatata 54360 ctccctttca ggctcctgga gtcttgtttacttctccctt tcactcccag atgcaggctg 54420 tttagaagcc caacccttag gagacagccagaaatgggag attttgcctg ttctctctgt 54480 attgagccaa ggggtggtgg gggcgggtagccactggcat tgctcaaagg cctatttaaa 54540 accacctctg tgttcactgt ggtctagggagactcctgat tgcagagctc catctactcc 54600 tggagctagg tgatttagga gccagacccttaggtacgag ctgtaaacat tggggtgctt 54660 gatgcataga caaattattc ccaggggtatgttcagacct ggttttatcc gtggggcgag 54720 ccaggggaag gaggcatggg aagtgcccttactgcttttc agccttcctg taagtctgtc 54780 gtttccctgc tccttctgct tcccagtgcaggctagttag aagcccaaca ctcagtcagc 54840 aactgataaa gtgggtagac gaagcccttccagggagaaa ctgggagctg ttagctaatt 54900 tttcagtact gaagtacttc tttcaaacgcattgctaatt tcaggagatg tttaacataa 54960 atacatcagc taagaagtct tactaatctaattgcatcag agctaaaaaa ttttgtgcaa 55020 atttaattct aagatttcca gaaaatgggcataaggacct aatacaacca aggactgcac 55080 agattgcctg taagagatcc ctcactggttagcaatcctg agttaaatac agattcagtc 55140 aggcccacta tacacaatag tagtagaatttaaaattata aagcagccct cagtgaaata 55200 gcattttaga gaagagaact taagaacaatctcaaactgc atgttaaatt tataactata 55260 ttgtctgtaa aagattatgc tacaattctgatatactaca attaaaaaca gttggaagaa 55320 aaggacttat attcccatct caatccttgattatactctc cattattggt atttctattg 55380 agtgttttta agtcatggca gtagaatcattcctagggat tctctcccta gaaaggatgt 55440 atttaactgc ttactttctg ttcttcacttacactcctct ccagctcagc aggtgtggct 55500 gaggtaggag taacagtccc tgacaccatcaccgagtgga aggcaggggc cttctgcctg 55560 tctgaagatg ctggacttgg tatctcttccactgcctctc tccgagcctt ccagcccttc 55620 tttgtggagc tcacaatgcc ttactctgtgattcgtggag aggccttcac actcaaggcc 55680 acggtcctaa actaccttcc caaatgcatccgggtaagga tctctttcct aaattaaata 55740 caaggtagcc atcaagtaaa ttaaaagttgcattcctagg aatactgcaa actcttgtat 55800 gcaaaatatg cttactagat attcatgattgtaaaagtta caggttttga gatctccaaa 55860 taccaaattg caccataaag caagtttctagcccttataa cccttcaaag ttagtatttg 55920 tgtggtatga agaattctga caggggtgacaaaagtcagt actctattct catgacagat 55980 tctacaaggt ttcaacctct acgatctcatatatttaact tttcgtagct cattcattat 56040 attaaaccta attttaaaag tcgtttgtgagcatcttacc tttgctgaaa ccataactgt 56100 ttataagtct tgtatcctct gccgggagatccgggggaat ggtcaaagtt ccagaccaaa 56160 gaggtagagc agcatgccat cattacctttcccttcctct ggtcccatca tgtgaaagag 56220 caggttgctt ccaaaataac tcagatttacctgtgtaaat ctgatacatt aagatccact 56280 taaatatatt tcaggtactt gatcttcatttatatcatct ttaatgtgag gcaacctgaa 56340 atctaaccaa ttcctcaaga tgctttacttcaaacccatt ctccctctgt tctccttccc 56400 tctctactcc ctttcttatg tgtggtttcaggtcagtgtg cagctggaag cctctcccgc 56460 cttcctagct gtcccagtgg agaaggaacaagcgcctcac tgcatctgtg caaacgggcg 56520 gcaaactgtg tcctgggcag taaccccaaagtcattaggt gagcaaaaaa ctgctagaga 56580 taattctcta ctcaaagatt gtatatggcagtgggaacct tatattgagt gctacttcct 56640 tcaggaaaag accactagat gctgcgatttttttcctttg ccttttattc taagatgcct 56700 acaaggatat cctcaacatc tccaccttgaattctcagta tcattcacct ctcatttgca 56760 tgtttccgtt cctgcttctg tgttttaataaaacaaaagt ttacagagca ttgaacattt 56820 ctaaatcttg agtttggagg catggaggaaggggaagatg ctattcattt ctactggcct 56880 tttttttcag gaaatgtgaa tttcactgtgagcgcagagg cactagagtc tcaagagctg 56940 tgtgggactg aggtgccttc agttcctgaacacggaagga aagacacagt catcaagcct 57000 ctgttggttg aagtaagtaa acctaaataatatatagtcc acaataatat ataatatatg 57060 tgggtaatat aataatatat ggatattttataatattatt ctcatgtatc tctctgtcct 57120 atctctctct tgatttactt tctgttttgttgggggtttt tgtttttgtt tttgaggcag 57180 agtcttgctc tgtcatccag gctggagtgcagtggcagga tctctgctca ctgcaacctc 57240 cgcctcctgg gttcaagcaa ttctcgtgcctcagcctcct gagtagctgg gattacaggt 57300 gtgcaccacc acgcccagct agtttttgtatttttagtag agacaggatt tcaccacgtt 57360 ggccaggctg gtctcgaact cctggcctcaagtgatctgc ccacctcagc ttcccaaagt 57420 gctgggatta taggtgtgag ccaccatgcacagcctccct ttgatttact ttcttaattt 57480 ttccttcatt tgttcatgca tcgaactacctcctacgtat attgcttata tgtacagaat 57540 tttcttagat aatacagttc aaatccttctcttcactatc caaatatctg tggtccctcc 57600 attaaaacac atgttctgaa ggtcagtccattctcactag cttttctttc ttttacctaa 57660 agcctgaagg actagagaag gaaacaacattcaactccct actttgtcca tcaggtaaga 57720 gtcaaccatc ataatttaaa aaacattaaagtctaacatt taaagttcaa agaacattta 57780 tatattattc ctacactttc tctgtgatctaagacctgaa gcaccatcaa tgcatttgac 57840 aaatgtggaa aatagttctt aggaaggccaagtaatttga tcagaatatc cctaggcctg 57900 cattctgagt cttgatcttt tgcagcacctgtgcaaacac caaatgactt tctgaccagt 57960 gtatggtatg ggcataggta gaaagtgggtagaatcaaaa ttaatattac caaaagggat 58020 gtttccttaa ataattaata atgcaaactatggacggctg aatttagggc attctaacac 58080 tgagttttac atagccaaca gtatttgataacgggattgc tatttcccaa aggaaaagtt 58140 gtcatggcct ttaccattat tgtcatattaatatctgttt gatgcctatc ccgtacctaa 58200 tgccctatca aacatttgag aaggaactgaagaaacttac aggaaaaatt taatacacta 58260 agaaatttat cagcacaatg cattctcaccccaaaccaac attgaatcaa catcatacat 58320 aggttcattg cctttctctg actacctacaaatttagtat gtttttcgta ctaaatactt 58380 tatctattca tctgttgcca agatgtaacacataaaatgt accctaaaaa cataacttcc 58440 ttgtcattta gccttatttc tacatttaagtgaactgatt acctatcatt caatcctttt 58500 atcatgactt ctccgtttct gagttactcattttgatgta tctcttaagt gtaagggcta 58560 atcatcaaat agttttacta aatttcattttaattaccaa cataatcaaa tgtgcctacc 58620 taattttaca aaaatatatt cttctttaaaaaaaaaaaca gaacatcaca ttaaaggtta 58680 atgtcacccc cctgaacatt tttcagtactttgccatcca tttatcttta gaaataatgt 58740 gtgtagatgt atatgtttgt ggatgtgtgatttacatata ataaactgta taagtttcat 58800 tctataaatc actgtttgtt tttcactcagcatcctgtct tggagattta cctatgttaa 58860 attgtagatc taggtctttc cttggaattgcttttaagcc tataatataa atacatcaca 58920 attctgctta gtgttttagt cttcctatattggttttttc aactattcac tagctttaaa 58980 aaattagtta gttaattata ataagagcctcttaatgaac atatgcaagt acagctaggg 59040 tagatccaaa atgttaaatt cctgagtcagagagcatatg catagatatg catagttttg 59100 tttggttggt tgttgttgtt gttctttaattacattgtaa actgaccatt tataattgta 59160 tatatataca gcatacaaag tgatgttatgatttatgaat aaaatgtgaa ataattaaat 59220 caagctaacc tgaaatactt atgttttgtggtgggaacat ttgaaattct ctaagcaagt 59280 ttgaaacata aaatacacta ttattaactatattcaccat gctgtgcaat agatcccaaa 59340 aagaaaaaaa tgtattcctt ctgtctgagactttgtgtcc cttgaacacc accttccttt 59400 tactccagct tcatcctcca taaccaccattctactctct gctcctgtga atttgaatgt 59460 tttagcttcc acatacaaat gagaacatgcaatatttgtt ttcctatacc tggcttattt 59520 cacataacat aatctcctcc agatttaatcatgctgccat aaatagcaga atgttcttgt 59580 tttttaaaat ggaatggaat tctatgtgtatataccaaat tttctttatc tgttcatctg 59640 ttgatgacac ttatgattcc ataactagacatcagtaatt tgttagggat tacattcaat 59700 atgtagattg ctttgggtag tgtggacattttaacagtat taattcttcc aatccatgaa 59760 cattgtattt tttttcattt atttgtgttctctttgattt ctttcatcag tgttttataa 59820 ttttcactgt acatttcacc tccttgattagatttatttc tacatattgt ttatagctat 59880 tgtaaatggg attgttttta tttctttctcaatcattcat tgttagtgaa cagaaaatac 59940 tactgatttt tatgtgttaa ttttgtatcttgcaacttta ttgcattcat ttataagttc 60000 ttgcagccct ttggtgaagt cttttgagcttccaatatat aagataatgt catcaccaac 60060 agtgaaaatt ttacttcttc cttgtcaatttggatatttt tcatttcttt ttcatgtttg 60120 attgctcttg ctgctacttc cagtgctactttgaaaataa atggtggcag tgggtatcct 60180 tgtcttgttc cagatcttaa aggaaagtctttcaattttc cactgttaaa tatgtaagct 60240 ataggtttat catacatgcc ctttattgtgttgaggaaca ttgcttgtat atctaatttg 60300 gtgagagttt tatcataaaa gagcattgaattctgtcaaa tactttttct ccatctaaca 60360 agatgatggt atggttttta cccttcattctgtaaatgta atgtatcaca tttattgata 60420 tgcatatgtt gaacaatttt tgcatctcagggataaatcc cacttgacta ggtagatgat 60480 ccttttactg tattgttgga tttagtctgctagtttattt cgtgtggttg gttggttggt 60540 ttagtttttt aagtgatgag cttttgctgtgttccccagg ctggacttga actcctgagt 60600 tcaagcaatc ctaccacctc agccacccacaggtgtatgc caccatgctt agctgctatg 60660 ctagtatttt attgaggatt tttgcatctatattcatcaa gaatattggt ctgtaattct 60720 tttttgtaat gtctttttat gacattggtatcagggtaat gcttgcctca taaaatgagc 60780 ttgaaagtat tccttcctct tccagcttttggcagagttt gagaaggatt ggtattcatt 60840 ctttttaaat gataagtaga acctagcagtgaagccaaca gttattaggc ttttctttaa 60900 tggaaaactt tttattactg attcaatctctttactcatt atttgtcagt tcagattttc 60960 tatttcttca tgattcagtc ttggtagtatgtatatgtct aggaatgcat tcatttcttc 61020 tagatcatcc aatttattgg tgtatacttatgcataatag tctcttatga tcctttgtat 61080 ttctgtggta tcagccataa tttctctttcatttctgatt ttatatattt aaggcctccc 61140 tcttttttct tagctaacct agctaaaagtttttgtctgt ctttttaaaa aaacaattca 61200 gtttcattga tcttttgtat tctttttctagtctctattt gatttatttc tgctctaatc 61260 tttattattt tcctttcttc tgccaattttgagcttactt tgttcttctt ttcctacttc 61320 tctgaggaat atcattagta tctttattggaaatttttct tcttttttgg tgtttattgt 61380 tataagcttt cctcttagaa ttgcttttactttatgctat gttttgttat gctccatttc 61440 catgttcatt tgtcttaaga tatttttgaatttcctttta aatttcttta ttgacccatt 61500 ggctgttcag gagcatgtca tttaattttcatatatttgt gaattttttc taattcctcc 61560 tgttactcat ttctagtttt catagtattgtggtcagaaa agatacttga tacgatgaaa 61620 cgatttcagt cttcttaaat ttgctaagacttgttttgtg ggctaaaata tgatctatct 61680 tggagaatgt ttcttgtgtg cttgagatgaaatgttctgt atgtatccat taggtctatt 61740 tgatctaaag tgttggtcaa gttcaacgttttcttattaa ttttctttct ggataatcta 61800 tccattttta aaagtgagat gttgaaattccctgatatta ctgcattgca acatatctct 61860 cccttcaacc tttaatattt gttttatatatttaggtgct ccaatgttgg atatgtatag 61920 atttacaatt gttatatcct tttgatgaattgaccctttt atcattatat aatgttctcc 61980 tttgtctctt tgtacagttt ttgactttaagtctgttttg ttgaatataa gtatagctac 62040 ccctgctctc ttttagtccc catttacctagaacatcttt ttccttctct tcactttcag 62100 tctatgtgtg tccttaaaaa ttaggtgagtctcttgtaat tagcatatgt ttcggtcctg 62160 tttttttaaa tccattcagt cactttatgtcttttaaatg gggaatttag tccatttgca 62220 ttcaaggtaa ttattaattt aaaaatgacttggtactacc attttgttgt tttctggttg 62280 ttttgcttct ttgttcctct tttgctgtcttcctttgtgg tctgatgttc tgtagtggta 62340 tgatttgaat cttttaaaat ttttgttctgtgcttctatt aaagattttt gccttgctgt 62400 tactatgggg tttacagtca atttcaagctgataacaact taactttgca ttctttcact 62460 cccccacaca cattttatgt cgttgatgtcagaatttaca tattttgtaa tgtgtattta 62520 ttgacaattt atttttagct atgcttgttattaatatttt gtcttttaac ccttgtacta 62580 gagataaaat tgctttaaat accatcattacagtcataga gtattttgaa tatggctcta 62640 tattacttat accattaaat tttgtgcttttgtgtttttg tattattaat taggggcctt 62700 ttgtttcagc ttaaagaact cccttcagtaattcctgaag gcaggcctaa tgttgacgac 62760 tcccttagct tttagtttgt ctgggaatgttttatttctc cctcatttct gaaagacagc 62820 tttgctggat gaagaattct tgattccatgttgtttttat ttttgttttc cttcagtact 62880 ttgaatatat tattccactc tccctcagcctgccgggtta ctgctaaaaa tccatggata 62940 gttgtattgg aattcctttg tatgtgatatgtttctttat caccttctgc ttttcagaat 63000 ttttttttgt ctttgatttt tgatagtttaattattgtgt cttagtgagc agttctttca 63060 tttgaatttc actggagacc tctgtgcctcctgtacttgg atgctagcat ctatccccta 63120 attagggaag ttttcagccc ttactgcttttttttttttc tgattctata ctcttttttt 63180 tcattattgc tttaaatgtg ctttatagtctctttcttct tcttctggac tttctttaat 63240 gcaaaggttt gatttcatga tgatgtcccataatttccat aggctttctt cattcttttg 63300 tctttctgct cttctgcctg gataattccaaatactctat ctttgagctc actgattctt 63360 ctgcttgatc aagtctgctg ttgagcttactttgaatttt taattttagt cattgtattc 63420 tttatttcca ggatttctat ttggtttctttttgattgtt tctatttatt ttttatttta 63480 caatattagc taagttgcag ataattgtttctatttcttt tttttttata ctttaagttc 63540 tagggtacat gtgcacaatg tgcaggtttattacatatgt atacatgtgc catgttggtg 63600 tgctgcaccc attaactcgt catttacattagctatatct cctaatgcta tccctccccc 63660 atcccctcac cccacaacag gccccggtgtgtgatgttcc ccttcctgtg tccaagtgtt 63720 ctcattgttc aattcccacc tatgagtgagaacatgcggt gtttgttttt ctgtccttgc 63780 aatagtttgc tgagaatgat ggtttccagcttcatccatg tcccaggaaa caacaggtgc 63840 tggagaggat gtggagaaat aggaacacttttacactgtt ggtgggacta taaactagtt 63900 caaccactgt ggaagtcaat gtggcgattcctcagggatc tagaactaga aatacatttg 63960 acccagccat cccattactg ggtatatacccaaaggatta taaatcatgc tgctataaag 64020 acacatgcac acatatgttt attgtggcactattcacaat cgtttctatg tcaaacttct 64080 cagtttgttg gtgtattgtt ttgcaaatttcatttaattt tgtatttata tattcttgta 64140 gtccactgaa tttcttcaag aggattattctgaattcttt gtcagtgatt tcatagatct 64200 ttatttctat gaggtcaatt ttttgagcttggccagtttc ttttggaggt gtcatcattc 64260 cttgattctt cataatcctg tgtccttgcattatttgtgc atttgaggag aaagccactt 64320 cttctggttt ttataggtat tctttggcagggataaaggt ttgctattta gtctagccta 64380 taattctgga aagatcagtt ggtgacaaccttgagcaggc agagttttca tgggttccct 64440 agttggctgg gccactgcct ttgctcttatgtttggtagg gccactggtt gggccttgct 64500 ctctggcaag atcactgttt tgtctctgctatctggtgga gctgctggct gggtactaca 64560 atggcctctg gtcaggccag tcacaagatgtgttgcctgg ctggatgatt ctgctatttg 64620 ggacctgaag ttaggcaggg tcacaatccgggctgtgagg ttaggtagag ttgttgcttg 64680 ggatgggcag aaataaatac tatacttcttagatgtgcat aatagaggat tgctacccca 64740 cccttgtgaa tggagccatg gagtgaggttttggctgagt tgagctaccc tttagactcc 64800 caggtcaagc atatttaacc cctacacttctatgaaatac acagaggtgg tgtctgctac 64860 ctgggtgggg tcactggcat aacctctgaagctgggctta cagactggcc atctggaaac 64920 tcaagctagg ttgaacttcc caacatgcttctgaaagtga ccagctcagt tttgcagatg 64980 ggctatgcag ttggctggta tctctgaatgggtgccatag ctggcagaaa cacagaagca 65040 ctaccaaaat ccacatgctg gtcactgtgagctctgtcct tctttgtttc tacctgacct 65100 cattacttcc tgtgttcccg gtgaaatgagaccagagtgg gcttcctgag aagtgtcttt 65160 gaatacttga gaatcttgat gtctacccctggttctcttc cccgctgtag aaactgtgac 65220 cccagggaac tcctctctat ctggcattgtgctaacctaa aggagtggga acaatgacat 65280 ggtcaaagtg agaccattct tcctactcttctaatttgtc ttcactcagt tctatgaaca 65340 atgtaggtgt cctagacttg tttccaagtattggggtttt caaaatagat tttctgatct 65400 gtggatagca gctagttgga ctttctgtggagggaggaag atcctgagac tttctagtcc 65460 atcatcttgc tttattctga attttaattgaactacaaaa cgaaaatcct cctctttatt 65520 acctaaatgc atttatactt ccaccaggatacatttccat agtgttatat ttgccatcat 65580 ctgttaccat caaagttttt aattttaatttttgccaaaa atttagaaaa aaaaattttg 65640 ctgttgtttt aatttatatt ttcttaattacaaggatgac cttatttttg catgtttatt 65700 aattgccttt ataatctttg gctatttgtcttttgagtag tttttttctg actcagttgt 65760 atgacactaa ttctttatct gttgaatatgttgcagatat tttctttcac ttggtcattt 65820 ttttaacttt gtttatggca tctgtttttttacaaaagtt ttaacattaa ttttatgaga 65880 aagggaagat aactgctaca tttttcatttgtatataatt caccaatact aaaattgtag 65940 taaatgtatg ttcatcagta gcagttattttattttcagt gagtcaagca ttttattttg 66000 cttagccatt tgtcctttaa ctatgcttatggcttttttt taagaaacat tttaatatga 66060 attttatgag gaagggattc agtatgaaaataaataccat acttctctca ttttcatttc 66120 atataattta ccaagattaa aatggaagtaaatgtatgtt catgagtagt aattatttta 66180 ttttcaatgc atcaaatact gttcgtcttcacttccttac cctcaatttt ctaggttttc 66240 cataaaaata ctatctttgt atatgaaatttgaagaaaga acatagcatt attatagaat 66300 tcaggacctt ttgtgggtaa ttttacttatgtatacttat agggctttgt tgttggtgtt 66360 ttctccatac aactgttgag taaggaagttggtggtggga actaaataga tcatcttgtg 66420 ataaccgtct tgtgtcagcc atcagatgacagcaactgaa tcacaacatc accaggctct 66480 tacaatttgt tgtcttattt ggcatgcgattctacataaa ttactgaaaa gatcattgaa 66540 gaagaaattc tgaaaatcac aggaaaccagtagcccattt ttaagatatt tatatattac 66600 tgttgtatta aaggcggaca acttttcaggaggagtttag gtataaggca tagtcctagc 66660 ttctgggtca tagagctgtt tagaaagatataatgcagaa ataattttca tatgtctgat 66720 ttgcttattt ctctaggtgg tgaggtttctgaagaattat ccctgaaact gccaccaaat 66780 gtggtagaag aatctgcccg agcttctgtctcagttttgg gtgagtctcc agcccctagt 66840 ggatccgggc attaacagct tctattatactatttttatt tcccataaat atttactaaa 66900 aataatacta taattttaac ttctttcttctcttcttctt tgggcttgtt tattgctttc 66960 aatcatactt ctatccctgg aagaatcatccttcctaaaa attctcaatt tctaagctca 67020 actaattatt tctgcttaat gactttgatagatgataatc tccaagcttt atgacttccc 67080 atctctccca ttctctagga gacatattaggctctgccat gcaaaacaca caaaatcttc 67140 tccagatgcc ctatggctgt ggagagcagaatatggtcct ctttgctcct aacatctatg 67200 tactggatta tctaaatgaa acacagcagcttactccaga gatcaagtcc aaggccattg 67260 gctatctcaa cactggtgag tgattacttgagtaagggaa aacttgaatg ttatttcaac 67320 tggatttccc agtaggtttc agttacttatgaatattatg atacattagc ttagctcact 67380 atgatagctg ctatgatagt taatttcaaggaaactatcc actctccaac ctccaataaa 67440 atatttaagg ctcagaaact cctaatctatgacaacaaaa tttaagaaat gtcacaagag 67500 aagccaaggt acttttagta atttctccaccctcagcatg cacattaatc cattgtgctg 67560 tttcgttaat cttcctttcc aggttaccagagacagttga actacaaaca ctatgatggc 67620 tcctacagca cctttgggga gcgatatggcaggaaccagg gcaacacctg gtaaggaaag 67680 aacaattttt tgagcttctt tttgtgtgccagctctttta catgtattac ctcaattata 67740 ttcacagcaa cactatcaga tatgtattatcagaccgatg gtttgttata ctagataaat 67800 ccaccaagat tagcaaggta atcagaagaaaacctgatat ccaaatacat gttatgttag 67860 gcttgtttcc aaaatggatc ctattaataatgtaccaagg ttttctttct gaaatggcta 67920 ttctttctaa agtagctacc ataaccatgagttttaaaat gatattgcca gtgaacatat 67980 ataacttcca gataaaccat gttaacttcagcttatattg tcacattcta agtcattcag 68040 cttgacttgg aatgaattca ttaataagaggaaacaattg agaaggaaac agtaatataa 68100 aacatttttt taaatcccta aagtaaagcaatattaaaat ttactgcatg taagagctgc 68160 atgtgagaag attctgtcat ctgcagaaggaaatctctaa agataagaga gatttaaagc 68220 cttactcaag taactaacaa aaataagtacattcaaatta cttgaatgta aatttgttca 68280 accattgtgg aagacagtat ggcgattcttcaaggatcta gaaccagaaa taccatttga 68340 cctagtaatc ccattactgg gtatatacccaaaggaatat aaatcattct actataatga 68400 cacatgcaca tgtatgttta tcgcggcactatttacaata acaaagtcat ggaactaacc 68460 caaatgctca tcaatgacag actggataaagaaaatgtgg tacatataca tcatggaata 68520 ctatgcagca ataaaaagaa atgaaatcatgtcctttgca gggacatgga tgaagctgga 68580 agccatcagc ctcagcaaac taacacaggaacagaaaacc aaacaccaca tttctcactc 68640 ataagtggga gttaagcaat gagaacacacggacacaggg acaggaacaa cacacaccag 68700 ggcctgttgg gaggtgtggg gtgacgggagggaactaagc ggatgggtca ataggtgcaa 68760 gaaaccacca tggcacacgt atacttatgtaacaaacctg cacgttctgc acatgtatct 68820 cggaactaaa ataaaattaa atatactaagactccctgtg gcaaagagag agttagcaag 68880 gaaatactac atctagcaga ttaatcaggcagactaaaga ttaatcaagg agataagctc 68940 tctaagtaca caagaatttt gttagctaactcacatcata tgaagcctgt tgctgtgaag 69000 tggttataaa accattttga caacataaacatcatgattg cttcctccct ggtcaggctc 69060 acagcctttg ttctgaagac ttttgcccaagctcgagcct acatcttcat cgatgaagca 69120 cacattaccc aagccctcat atggctctcccagaggcaga aggacaatgg ctgtttcagg 69180 agctctgggt cactgctcaa caatgccataaaggtgaatc attctggagc tagttttgat 69240 ttgtccatta tgatatctgc aaggatgaggataggaagtg ataatgtgaa aaattctaag 69300 ggaaagcctc agaggaaaat aaaacctggatggcaccaaa aaagagggga tagaacaaaa 69360 gttgattgtg atactttgcc ctatagggatggatatgggt aaggatgaat tccatgacac 69420 agcagaatag aaagaactaa tcaatagcattctcagaagt tgaattattc agatctctct 69480 ctcgtattca cagggaggag tagaagatgaagtgaccctc tccgcctata tcaccatcgc 69540 ccttctggag attcctctca cagtcactgtaggtaccacc ccattcctct gctgaaggag 69600 agttctggat gcaatgaaac tgctgacctgctgtctgaaa tactatccta ttaaaagcaa 69660 agcatcagct ttctttctat gcaatgccagtgcttcccag atctacagag aatttggtca 69720 gcccattaag aaaggtttaa attttcccagtaattcccct aggctattta ccaccaccac 69780 tcaaaaaaga atcttaaaga tgtatcttttgaatgtgaga ataacagata aaaataatat 69840 tatatctatt gataagaatg aggaatcgttggaaaaatgc gtttgaaaaa cttctgtgct 69900 gtgatccgtg tatttgcctg ggaatgctaatatgcctgtt tacatagctt agttcccttc 69960 ttgttctgcc ttcacagcac cctgttgtccgcaatgccct gttttgcctg gagtcagcct 70020 ggaagacagc acaagaaggg gaccatggcagccatgtata taccaaagca ctgctggcct 70080 atgcttttgc cctggcaggt aaccaggacaagaggaagga agtactcaag tcacttaatg 70140 aggaagctgt gaagaaaggt gagagcacacctgagatcct tctcctggcc catcctctgt 70200 atcaagaact gcatggcaaa aatccctcactcctacctcc tgtgatccct gtctcctctc 70260 ttcttttcta tatatcatat atattttgtccatattgcat cttataaaat ctaggatttc 70320 ttaatcaaat cagaaatcag aagacaagaggccgtgcaga tgcttctcaa ttacgatggg 70380 gttatatcct gacaaactca ttgtaaagtctaaaaaatct taagtgggac cattgtaagt 70440 cagggaccat ctctatagta tgctggtaagaagagcattc tctggagact agctccaaaa 70500 tgtgctacct atgtgagctt gggaaagtcattaacttcct tgtgtttcag ttccttcatc 70560 agtaaaatgg ggataataat agtatttacctcacagagct gttgtaataa atgaattggt 70620 acacgtaaaa cacttagtag agtacatgtcacatagcaaa tcctataaaa gtactagtta 70680 ttacaattaa catatcagtt ctcaatatatgcccaaccct tacctggtac attatataac 70740 cttaaacata agaaaataat catggaagtaactccttgaa tgaattctgg tattttaagc 70800 ccatttcata agaccaataa tgttgaccaatctactcata ttcacacagt acttctacat 70860 ataccatggt ctatatgagc ggttgaagaaatagaaaata aaatgcaaat cacaagatgt 70920 ccattaaaac agtctacctt tttcctttgacagccattaa ttcttcttaa aatgtattga 70980 gaaatatttt ataatagata tacaaaagggcataagctat aactagaaaa cactgtacaa 71040 ctctctcata gattaagaaa tagaaaattaccgacatggg aaaaataaat cccttgtgta 71100 tcactaccac ctccagaggc aatcattatcctgaatttgt cgttacaatt ccatggattt 71160 ccttatattt ttgctgcata tgtatccctaactaatattt agaatcttca catgtgtttc 71220 atcttgagag aaacaagatt tttatttctctcttattcaa gaaacaagag aaacattttt 71280 gaatatttca gcagcttagt tttttgtttgtctgcttttt attttctaag ttcaacacta 71340 tgttgatgaa gacccctata caagtatgtgcagagtcagc tcattaattt tcactgctgc 71400 ataatatatt acagtctata aattagtcataatttacaca tcgagtttct cctcatggat 71460 tttttttatg ttttgctatt aaaaaaaatgctgcaatgaa tactcatgtg cctgttttct 71520 tgtgcatctg tgtttctcca aattatgctttgagaagcat aatgactgat tagtgggcta 71580 agcacatctt ccccattgct gaatattgccaaagagcagt tggcttccca cagcagtgta 71640 tattggttcc cattgtttca catccatgtcagcctttggt atttcaagga ttactgtatt 71700 ttttttttca atttaatgag tagaaactctactatttcat gtgatctgtg atcctcacaa 71760 gaaactgata aagacacact ttataggaaatgtaacaaac tccagctata gtctaatata 71820 acatcataaa taggaaatag ccagactcaatgagacaact gctgtgccat ttctttctcc 71880 caccaacccg actatagcaa cgatttgaaaacatagatag gcataggctt ctgactccag 71940 catcaatatc tgccttagct gggctaaaacacaccaaatt cagatttaca tgaagggaaa 72000 agcatctaca tacagtacag gggattataatgggcatgca attctcattt cagcactggc 72060 ttgggtactt tcaccttgaa ttaaatataaatatgtaggc acttataaat atctttttct 72120 catctttaag acaactctgt ccattgggagcgccctcaga aacccaaggc accagtgggg 72180 catttttacg aaccccaggc tccctctgctgaggtggaga tgacatccta tgtgctcctc 72240 gcttatctca cggcccagcc agccccaacctcggaggacc tgacctctgc aaccaacatc 72300 gtgaagtgga tcacgaagca gcagaatgcccagggcggtt tctcctccac ccaggttggt 72360 gatttgccaa aaccttttat ttcaccttcaggtagcaaaa gatttgaatg aaaaagaaac 72420 aaacacatcc aagaagaaaa aaatacagatgacagtaact tgaaatgagg aaaagttttc 72480 agtatccaag gataatggaa ataaaagcaaatcaaagtca aagagggcca aaaggaaatg 72540 ctcagaatcc cggcacccca tcgctgtgttattatccatc tcctatttcc cataacaaca 72600 ctgccttcct caagcagcag tggagcaccagcagaatgaa ggagatgtct cctgccattc 72660 tcctgaaagc tctagggtct ctttcaaactgttcaaagga actctactca aaatccaaca 72720 acctctcctc gcaaatctct ccattcttaggtccccttta ataggctttt ctcaaaacta 72780 cacattttgt gcttccccta ttcacctttttttttttttt ttttttaaga cagagtcttg 72840 ctctgtcacc taggctggaa tgcagtggtgcaatctcggc tcactgcaac ctccatctcc 72900 caggttcaag cgattctagt gcctcagcctcccaagtatc taggattaca gtcatgtgca 72960 atcatgcctg gctaattttt gtatttttagtagagacgag gttttgccat gttgcccagg 73020 ctgatctcga agtcctgagc tcaggcaatccatccgcctt ggcctcacaa agtgctagga 73080 ttataggtgt gagccactgc gtccagccccctattcacct cttaatacac aaacatttat 73140 tcatcaggag cataaagaac tgtctttattcatccaacct cctaaatcta gctatataac 73200 catgtatctg aacaattcat tgatatgtacacagcagaaa gttttatctt cagagaattc 73260 ggatgtttgc ttatataccc taaaacggaaaaaatgtgac aaaatggcat tccatcctat 73320 ttccattgta ttaatctttt atcatatgaatgaaaaaaac taagtaattt tgttaaaggt 73380 tatcattcat ttattagaaa catattatttgaaggaggcc aagcaggttt aatgttgttg 73440 aggatacata ccagcagaca ttcactgggaacaggaaatc atccaataaa aagggaaagc 73500 caaataaaaa tgtcattaaa tccagaagataattataata ctcatctttt atttcttttg 73560 gagaaactga agcatgactc tgctcatggctgcaaagaac cttgggttct ctccaggaca 73620 ctgacctcag caactgagca aagtttaatatgggagagag ccagactgaa ctttgcttga 73680 gtggtggcag atatgagcat agttgtcaagaaagacatgt tagcaaatag ctgatgccaa 73740 taactgattg ccattcacat gttttccacattccatgtcc cacatatact tacagagaga 73800 aaaggatcaa ttttctgata aataaaataaacatgtaggg catacagtcc aaggtagata 73860 tgtgaatgtt atggttcttc aactatctaaagattataat caatcttgaa attacagctc 73920 ctatatttaa gtagtgaggg aagtaggaaatcaaagtccc tcacatgggt ctttgaaaaa 73980 tatctcagcc ctcaaagcct tataatgcccaatgggttct ctcactcatc tgtctctaac 74040 aggacacagt ggtggctctc catgctctgtccaaatatgg agcagccaca tttaccagga 74100 ctgggaaggc tgcacaggtg actatccagtcttcagggac attttccagc aaattccaag 74160 tggacaacaa caaccgcctg ttactgcagcaggtctcatt gccagagctg cctggggaat 74220 acagcatgaa agtgacagga gaaggatgtgtctacctcca ggtgagactc ttgggcaggt 74280 gaggacagga cagatgagga cagcagctgttctctctgag aagtcctaac tcagaaaaca 74340 atgggacaga tcagagaaag ggttagggacgtggacagga attctgggaa agggcaaaaa 74400 actgattttg tctttgatgt tctatagacatccttgaaat acaatattct cccagaaaag 74460 gaagagttcc cctttgcttt aggagtgcagactctgcctc aaacttgtga tgaacccaaa 74520 gcccacacca gcttccaaat ctccctaagtgtcaggtaag accttctgac tctatcacct 74580 aatcctaaga ataaccacca gtcttctttcgggaactcct ctttaagtaa agcagtgcaa 74640 cagtagatat ttgcactatt cacaaaaaatgcaatgtatt ctcttaagtt gatataattt 74700 ctcaatgatg gggtttacat tgtccatccaggatctacta ttgtgcaacc tcattgttta 74760 aagggtaata atttccctca ataacaactaagtaaatatt acccattgcc tctgacctga 74820 attccttgtt atgtaatgaa atcctatattattcttgctt tattgaagat agagatgaag 74880 aattattgaa aagtttgaat agaaggaagtagtgactcct tagttagaat tcctactggc 74940 aataataaat ctcaggttat atatgatataattaatttgg ggggaagata cacttatatg 75000 catcaatatt taaatagctg cagatctgataaaaaactct ctctccacaa acatattatt 75060 acttggttgg agatactatt caggaaaaaagttaggacaa aatacatgta acaaataact 75120 ggcacacatc aaaaagaatg agatcatgacctttgcagga acatggatgg agatggaggt 75180 cattatcctt ggcaaactag cacaggaatggaaaaccaaa cactgcatgt tctcatttgt 75240 aagtgggagc taaatgatga gaacatatggacacaaaaag gagaacaaca gacaccagag 75300 cctacttgag ggttaaggat gggaggagggagaagatcag aaaaaaacaa caattcagtg 75360 caaaatttag tacccaagtg ataaagtaatctgtacacca aacccccatg acacgagttt 75420 acctatataa caaacctgca tgtgtacgcctgaacctaaa agttaagtat atatatatat 75480 atttttttca tttaatttgg tgtatatatatgccaaaaaa taaattaagc agtccaaatt 75540 tcggatgcaa actctcgggg acaagacgctaggtgtttct aagtgttttg ttgaaagcca 75600 gtgtttaagt aaacattata aattattgttgtttttgtaa ataatgtaga ctgaaattta 75660 ttattcataa tatacatcat tttgtcagctgaaagaaaat aaaagtaaac aaataaataa 75720 aataactggc atagattaga ggtcacaaacagcctgtgca tcacttgtag agctttctta 75780 aaatgcagat cctcagccgg gcgtggtggctcacgcctgt aatctcagca ctttgggagg 75840 ccaaggcggg cagattacct taggtcgggagttcaagacc agcctgacca acatggagaa 75900 accccgtctg tactaaaaat acaaaattagtcggacgtgg tggtgcatgc ctgtaatccc 75960 agctactcgg gaggctgagg caggagaatcacttgaaccc aggaggcgga ggttgcggtg 76020 agccgaaatc atgccattgc actccagcctgggcaagaag agtgaaaaac tccatcaaaa 76080 aaaaaaaatg cagatcctca gcccccatacactagacatt ctgattcatc aggtctagag 76140 tagggcctgg tctctgtggc tttaacaggcttcctaaaaa ttctacgcac accactttta 76200 caaaccactg ggataagata tttgggaagacttacgtgta ccttttagag ctgtggaatg 76260 cttaacatgg acatagaaga agaaaatatttaaaaacaca gaaaacccta atcctttcct 76320 cccctggatc ctcagttaca cagggagccgctctgcctcc aacatggcga tcgttgatgt 76380 gaagatggtc tctggcttca ttcccctgaagccaacagtg aaaatggtag gtttatcata 76440 accccagact gccctatttt atttaatgatgtatgtatcc ccagcataag acaatactaa 76500 tatcaaaata ctattaaagt caatctctatcaaagcctta tcctttttcc agctcagaaa 76560 tataatcaca tgtgtttgta tgaatgctgaccatgtgcag agcactgtgc taggacccat 76620 gactacaaga aaaagattgt cagcaggttcctgcttttca atttcttctt agcttagaat 76680 tttgctaaga agataaaaga tatgaacacgaaccagtgaa aaatatgaaa atgactgatt 76740 ggcataaact ataagtatta cagaagttaaaagaaaaata gagcaagcaa aacaggaaaa 76800 aaactcctta tgaagaaata gaaactgaattgaactttga aatatgagta actaccaagt 76860 ttaggatact tagctgtctt ttcttcagataaataacttt acacattagt cgtgtgttat 76920 actaatagta aaccctttat gcctttcatttttaattgta ttacattata tatttcctta 76980 caaaaagcat ttgaagaatt ctaccctcagggttattttg gcaatacaaa gattttttct 77040 ctggatcccc caggggtttc atctatttattaacatttgt ggtatttcaa ttttcttcag 77100 cttgaaagat ctaaccatgt gagccggacagaagtcagca gcaaccatgt cttgatttac 77160 cttgataagg taagagaact tccagtctatttgcaaaaaa acgtagataa taatcctcta 77220 agggaacatc tgggaaggta aatgcattttagaaacatca cttccatgct agaaatttga 77280 gaattctaat gttaactcta aaagaatgttcttctctcct ttatttatat ttcaccaggg 77340 attacaggta gaaatggctt attatgatcttgggatatga atattcctaa aatcccataa 77400 gcaagaaatc ttcacaaaat gtgtttattatgttgacaag ttttttggat acccagtaat 77460 ataaggaagt agcccttgtg attagtcaattattagttaa ttatcaacat actcaacaac 77520 aatatgaaag ggaaaaaaaa ctgtcagtctccacaaggac ttgaaccata aaataataag 77580 accagttcac cagtaaacca atctgattttatagatatgt gtggtaggag agtttgttca 77640 tgcataagtt gatgggaatt atagtttacaaattttatga aacttaagcc tgggaagatc 77700 aaccttttag atgcctcttt gagtctacgcaagtattcct gcaagacaga gaagtcaaac 77760 tataccaaat ctctggatat taaaaaatgaacacagttag tcatccaata aaaagtatat 77820 atcatttacc cccatgaaca gagctatgtattggcattga cagaggtata tgcgttatgt 77880 tagttattta agaaataatc tggagaatttatcatcccct ctgagagatt tctgcacaat 77940 ttaattaagg accctatagt gtgctgtaggataataaagc ttttccccca aaaaacaggt 78000 gaatacttaa actaattcaa agagagaagaaagcttcctg aaaggtcatt taattgactt 78060 ttgctttcca ggtgtcaaat cagacactgagcttgttctt cacggttctg caagatgtcc 78120 cagtaagaga tctgaaacca gccatagtgaaagtctatga ttactacgag acgggtgagt 78180 gagagtgatt ttcacgtaga aatatttaattcctgatcac agaaattcag gtttaggaga 78240 tgtgttgggg ttatttatta cattaagtaattacattatc acttcatttt gtctccatca 78300 agtctgatgc ccctcttttt gtctcttatacatacattat agaaacaacc tacattataa 78360 atttatcaac tactaataca aaacacctgtgggatattta gttccctttt catcagataa 78420 atggactgta tgacaatatg agatttaagtaagtagaaca tctgaagagt ccttcaggag 78480 tttgggataa aagaatatat aaaacactatatttgaaagg agaatataag gtagcaagca 78540 acacatcaga tgaatgatgc ttatgtttctggtacaatac tgttcttccc acaacaaact 78600 ccttccttgg cctgtatccc acagatgtttgctttctttc tcacttcatg taatgatttc 78660 tggttttttg ttggtttttt ttttttcagatgagtttgca attgctgagt acaatgctcc 78720 ttgcagcaaa ggtaagccac tcacactcctccaaaaggca gtcagagctc cttcagcttg 78780 ccccccaaac cttctccttc ataaaacgctgggtaaatat ttgtcaaaaa catcaaatta 78840 ctcacactgc acattattat agaaaaacacatttattgga gagggccgct gactctgtca 78900 aacctcagag agtccatagg attgcttatgggtaatgatt tggaatagat ttggtttccc 78960 actgtactga ttaggtttcc ttgggcactatgctacccag aactaaggga aagaatactc 79020 tctgctcatg gagacccaaa tctgtcttaattttttttct ttccaatgtc acagatcttg 79080 gaaatgcttg aagaccacaa ggctgaaaagtgctttgctg gagtcctgtt ctcagagctc 79140 cacagaagac acgtgttttt gtatctttaaagacttgatg aataaacact ttttctggtc 79200 aatgtctttc cctgtttcct gttcattcaataaatatcat tgtacatttc catatgattc 79260 ccaatagaat accaagatta aacttaaaggaatcaagtgc tgaaggactt cagaatacaa 79320 aaaaatgata cagtgatgtc ggtctgagtaggcttcatgt aaggactgtg gggaaagaag 79380 aaagtattgg gttatgtact aggaaagtgtaaagtgtgtt tggttatggg aataccctat 79440 gaaaaaccca aagggtgaat ttttatgagaaaataaaaga ctgacttcac cagaaaagac 79500 tttttacatt aaaatgaagt agaatgaaatacaacattga acatgtcata ttgagaggca 79560 agataattgg gacttgacct gaattgggagtgatgtgtcc tatgttacac caaaatctgc 79620 cactgatgag agtgatcagt cagttaacctggggtttcag attcaataat agatgagctg 79680 aaaataatga agggaggatt catgcagaagcacgttttct cagaagaagg aatgtgtatg 79740 actcaaagtc caaataggag tattatattggatcatcttt cttctggaac tttgagccag 79800 gattaaagga tagctgtaaa gtcaaggagatattctgatg cagaaatcag ttctcacaac 79860 atctgattga tgtctgatgt ctcacaacatctctttagtc tatttttaaa atatataatt 79920 ttctttgcag taagtattgc gacatatatttccattctat agaaggggaa gcaaaacttc 79980 aggagttttt gaagtaggaa aggttaaagcaggaggattg agccaagaga gtctgaggac 80040 aatcgtagga gtcctactct tcatttggcacaaaaatgac aatgcttagt taggcagaag 80100 gtgagtatgg attgtataaa ctaagaactggaaaagactt tgcagttcaa ggaatcctta 80160 gctctgtctc caggctagac aaaataagaaataaaagcta tcacttctgt gtggtgctta 80220 tagaatagaa ttaacatatc agcattatgggatctttagg gtgtcgcttt cctggccagt 80280 ctagtggcac ctttgcctga gttttgctctgggcccactg ggctgcttct gcccactcgc 80340 acttgctacc aacctggatc ccggatccaagggagattga gacgggtgga gcagaggggt 80400 gtgctagggg tgtgtgagca agcgtggccactgtgcagtc acacacacaa gctgctgccc 80460 aggttgggca gctccaggtg ccagcacaggctctccatga ggtggctgga ccaggcacac 80520 aacaaacagc ttccccctgg accaggcgcatcacaagcag cttccaacag tggcactggc 80580 gaatgcagtg acgccaacca gggccccaaagagggagtca cagcccgggc tcaaggagct 80640 cccaggtctg ggcttctccg agggccagagctcttctctc cctgtgggga gcaaggggca 80700 tgttgcagcc ctgtttgtgt tacagctcttttaaccttgc tgtgcagctc ctcagctcct 80760 gcatcaagca gacaagtgga gaatgagacagatgaagagg agcattactg agcaatggaa 80820 cagaaggata aggaagatga agaggaaggagacctgcagt tagtagctca tttccacagt 80880 aaggatgtcc tttccacagc aagggtgtcccaacgagtgt tcagcttcta gcagaacgga 80940 gaccctggag tggctggctc ctctctgcaaacaggtcttc ccattgagtg ttcagctttc 81000 agcagagagg aggttctgga atgggtagtttctctccaca ggtaggtcat ccattgtctt 81060 cccatcctct cttccaatct agctgagtctgggggatttt atgagcctca gagggaggaa 81120 atgcatgctg attggtccat gggcagccatgagtgggccc agggagcagc accacaagtt 81180 acctctctgg tctgcaggct tcaagccctcaccagcttga gggtgggact tcactgggga 81240 cccatcccct tccacccagg aacctgtctgcctcctgctc ccaggctgtt catgccaagg 81300 agcgcctgca agtcagtgtc cagctgtcttcagacccctc tcagcctccc tcccacgctt 81360 gttggtgccc aagttccaaa gggggccgagacggcagggg gctggcgtat cagcactgtc 81420 ctgagcgtgt gcacgctcgg ccaggctgtgacagtaccca ggctcggccc gaccttgctc 81480 tgagttcaga gtgggtgcta acagtggagagaagccaggc agccggagta ggcaccctgg 81540 agcctgcagt gggcagggga ctttgctgggcctctgagag cacagaaaat gtccacagcc 81600 gcggcaaggt ggctgcagct gcaccctgggagctcctgct ccaccagttc ggaaggggcg 81660 gggctcctgc ttgtccctgg ctcacctgctcctgagtgtg caggtccggt ggcgcctcct 81720 tgcaggctgg gctgatgggc gggggcgggggggggggggg ggaggaaggg aatgttccag 81780 gtcctccctg ggcccgggac tgtgtccggggcagggatga cgtcgctgca agttcttccc 81840 gtggccccgg ggctcagggg cagcccaggactctccctcg cccggctcac ggccctgcct 81900 ggggggcgcc tccgggagca gatcacgagccctggggctc agccctcagg cgcgtctagc 81960 tcggcggtca ccccagtgcc gggaggaccctgaagacgcg ccccaggcgg ccctactcag 82020 agcctcctcc caaggcccag gaatgcggcgctgtcggagg tgtgcgcggt ggccacaccg 82080 ctgtccgggt ccccaaagcg ggccccgctcccacttctcg ccttggcccc gaaccctggg 82140 tccagcccca gcgctttgtg tgcgaacaccgctccgcccc ggacccagct ccgccttggg 82200 gcccctctct gcctgcccct ccgtgcccgactacactgct tcccctccgg cgggcgactc 82260 agcccggtcc atcgtggcgg cttccagggcggcaggctcc gggagtactc ccggggccgg 82320 ctccaaggac tgttcccctc ctccccactcccactccgcg gcggcggcgg gcgagagcgg 82380 cgacataggg ccagggtccg gagcggtggaggctcctggc cggggagcac gtcgccccac 82440 ccggcaacgc gaggatggtg gcggcgcagtcggctgcttt ggggtctcaa ggcacagggg 82500 acgcgaggca cagatgtccc acagcagccactgcggctcc cgcagctgct ccgccgccgc 82560 tgcccgcccc tccctgctgc agctggcgtgatggcagcgg cagctctgga cgccccactg 82620 ctgccaccat cagccttgtg aaataggtactaccttaaca aatgaaattg aagcagagac 82680 atgtaatttg cccaaagtta ctaagttagtgacaaagcta gaattcaaga ccaagaagtc 82740 tagcttccat gctcttaact tccaaccatggtgacacctc aaacaacttc agacaaaaag 82800 gccaggagaa agtatatttc agagcttaataaacattata attagctgtc aaattaagta 82860 tcaagccagg gcacagaaca taaaagaaatcagagtatgg ctatgggaac aagacaacag 82920 gattataatt ttacctcttg gttctagtttttttctttgt tcatatggaa atcgttactg 82980 aaaaggtact ttaaggatat gcttgttgcaaatcattagc tgtatcactg accaagagtg 83040 tttattcctg aaatactaac tgattgcctactatctgcca ggcacaatat cccatgctat 83100 aatacaaaat taaacaaaat aggattcctcccttagaaaa actcaccgca gagtaaaaga 83160 aaaagataca catctgggtc attataatgatcaggtgctc aagctattca ttgccccagt 83220 ggactggaga tacaatggcc ttctcaagttttggggtatt acactcaaat tcatatgtaa 83280 tactggagaa aaggcctaat tacaacttaaactggatttc ccaccagcct ggtggcatgg 83340 gaatcttgga attaaaatta cgtagaatttttaaaagtga tgtatcttct acatctgatt 83400 ttgtgaactg aagtttattc tttccaggaaagcatataga tacacgacag gaaatgaaat 83460 ggatacttgt tggggtcagt tttatgtatagtttgtattt tattttgaaa tatgatacac 83520 tgctattctc ttgcattttc ttatatgtgactcaccacta accctatatt cccccatttc 83580 aggccagttg gtcataagca tccatttgcctcagagaata ctgggttgtt atgacaagaa 83640 tataaagttg gaaagaaata gaatatttgagtctaccctg taagaataaa aagaataaaa 83700 ggggtttaaa tttatttaga ccctattgtttaatcaagaa ttctggccag gagcagtggt 83760 tcatgcctat aatcccaatg ctttaggaggccaaggcagg aggatcattt gaggccaaga 83820 gtttgagacc agcctgggca aattattgctcgggaaaaaa aggtcttatt tagtatttta 83880 gtctttacaa tgtttttttc tattatgcaatattctctca aatactttat gtccatccac 83940 gttgtctgag acatgccact ttaacattctagttatgctg tagctgtcat tttaccctaa 84000 gccgttagta gtactgatcc acataaaattgggctgttta ggtgtttaac tgtttaaatg 84060 tataatatat ctgatatatt tatatattgtataaaaaatc acctaacaca atagatattt 84120 actatgtctg ctataaatat atatcatataacatataaca atatattata aatgataata 84180 tactataata taaaatataa taaatatattataaatgtac aatatatccg atataaatat 84240 ataaatatgt caactatatt atacatataaatgtatatgt ataatatata catatatgta 84300 taatacaatg tatttattat gtatatatataaatgtatat gtataatata taaatgtata 84360 atatatctga tataaacata tcagatatattatccatcta ttgtgtcagg tgattcttta 84420 tacaatatat aaatatatca gatatattatccatctattg tgtcaggtga ttttttatac 84480 aatatataaa tatatcagat acattatccatctattgtgt caggtgattt tttatacaat 84540 atataaatat atcagatata ttatccatctattgtgtcag gtgatttttt atacaatata 84600 caaatatatc agatatatta tccatctattgtgtcaggtg attttttata caatatataa 84660 atatatcaga tacattatcc atctattgtgtcaggtgatt ttttatacaa tatataaata 84720 tatcagatat attatccatc tattgtgtcaggtgattttt tatacaatat ataaatatat 84780 cagatatatt atccatctat tgtgtcaggtgattttttat acaatatata aatatatcag 84840 atatattaca tatctattgt gtcaggtgatttcttatacg atatataaat atatcagata 84900 tattatccat ctattgtgtc aggtgattttttatacacta tataaatata tcagatatac 84960 tatacagttc agcccatcaa agcaccatattttggggggt tggtttccgt gtcccaacac 85020 tagctacgaa aatattagct attagctacctataactctt cagtagtaaa ttcaagaaac 85080 gtaaagtaat tctcttcatt aagttcttgccttgtctact aaaaaaatgg tcatcaccga 85140 tgtggacaat gaagacctgt ggggttaaaagctctaacta gtatgcctcc aagattcttt 85200 gattgcctgc catcatgatc gaagaataaataacttcttt ttcatcttat ttatttattt 85260 tttgtagaga tagggtctcg ctatgctgcccaggctggtc tcaaactcct gggctcaaga 85320 gatccttcta cttaagcctc tcaaagtgctggaattacag gggtgagtca ccacgactga 85380 ccattaataa tttctttcaa tgacactttaacataggctc attcatcttt acctctaaag 85440 aaaagtcttt ctggtctttt taaaattatattttttggcc aggcacaatg gccaggtgcg 85500 gtggctgaca cctgtaatcc tagcactttgggaggccaag gtaggaagat tgcttgaggc 85560 caggagtgca agaccaacct ggcaaacatctggaaaacat agcaaggccc catctctatt 85620 aaaaaaaaat taattctatt tttctaagagaaaaaagatt cccaattcaa caacactttt 85680 caaaaacttt atctggcagc tactcaggagattgagatgg gaggatcata tgaagcccag 85740 gaattcaaaa ccagtgtggg caacatagtgagatcctatc tcataaaaaa ataaaaaata 85800 aaaaaagctt tacttggaat acaacccatgactctggtta taaatacaaa attcttcaaa 85860 ttcatttaaa ggaatttaat cctagcttctcggatgaaaa aaggaaataa tattcacaat 85920 ttgatccatc atcagtagac aagttaaatgtgtttcacaa aagcaagaca tattaattaa 85980 gcaaaatcat attcgagtaa ccacaggaaatataaatata ctgtctctta cctagagaaa 86040 tcttatagtc taattgtgaa gatagtcttcacgtgacgaa aaagatcatc attaatccaa 86100 aacatataag ttataaagaa gcgacatataccagcaattc taaaatctgg ttagcatcct 86160 ttgtagaatt tattttaaaa tgcagatatccaggtctcat caataaagat ttaattaatt 86220 atttttgggg atgtgctcag acatctgcgttttttgtttt ttgttttcgt ttttttgttt 86280 tttgagatgg agtctcactc tgttgcccaggctggagtgc aatggcgcaa tctcagctca 86340 ctgcaacctc tgctcccagg ttcaagcaattcttctgcct cagcctccct aggagctgga 86400 actataggcg cccaccacca cgttgggctaacaggcatct atgtttttaa tgaactctgt 86460 aggtggttct atcatgcagt tagttttcagaaccattcac actgacagta aaggctattt 86520 attcccagca gttgaaagac cactaaggacacaggaatag ttagcaaagc tacttaaaga 86580 tgccagggct ggggccgggt gcgatggctcacgcctgtaa tcccagcact ttgggaggcc 86640 aaggtgggca gatcatgagg tcaggagatcgagaccatcc tggctaacac aatgaagccc 86700 cgtctctaca aacaaacaaa caaacaaacaaaatacaaaa aattagccgg atgtggtggc 86760 gggcacctgt agtcccaact actcgggaggctgaggcagc agaatggctt gaacccagga 86820 ggcggagctt gcagtgagcc gagatcacgccactgcactc cagcctgggc gacagagcga 86880 aactccatct caaaaaaaaa aaaaaaaaaaaaaaaaagat gccagggctg gctgggcaca 86940 gtggctcaca cctgtaaccc caacactttggtttgggagg ccaaggcgga tggattgctt 87000 gagttcaggg gttcaagacc agcccaggaaacatggcaaa acctcatctc taccaaaaac 87060 acaaaaatta gccgggcata gtggcatgcacctgtggtcc cagctactca ggaggctgag 87120 gtgggaggat agctggagcc tgggaagctgcagtgatcag tgatcatgtc accacactcc 87180 agcctcggtg acagagcaag aacctgtctcaacatacata catgcatata taaaattaaa 87240 cataaaaaca aaaataaata aagatgtcagggcttatgtt gaaccttaac tgagagcaag 87300 attcaaaaga cactgaggct tatttttctttcttatatct atagttacac agggagctgt 87360 ctaatcttgg atgtatccaa gttgatatctggttttatcc attgaaaccc acagtgaaaa 87420 tggtaaatag gtgctaggtg tttggatttttttaatccaa tgtaagaata aaacaatggt 87480 atcctaataa tgtcaaagca acattggtcataatctaagg aaattgaatt catatagtac 87540 caaatatata tttagcattg tgctaggtgctgatacattc tagataaaaa tattacacat 87600 gggtaaccaa aactgtcaaa tgacatttcagggcaagata taattaagta ccaaaatcat 87660 tggcatagtc tttaagtact gtgaaatgtagagaaagctg agatgaatgg cagtgtagag 87720 acagatggct ttgcaaatca tctcagatgactagctatcc aatgtgagga cacttctccc 87780 tcaccttcaa acaaatgcta aagacgcctgttacttaatc atatgaatat tcaatcttgt 87840 atctaatgtg gtggtattca taatactctgtatatgtttt catcttactg gacaagtgtc 87900 ttcgaactca tttaaatgaa tttaaccccagctttgttta tgtatagact tcttcaatct 87960 catagtctat ttgtctcttt gtaccccacaggctttcttt attcagtaac actggtgatt 88020 tctcttattt tccttagctt gaaagatctagccacgtgag caggacagaa gtgcacaaca 88080 accatatctt gatttctgtg gaccaggtggggcccctgcc agccttgcta gacagaccca 88140 ggtgaacagt ccttctaggg gatctcatcaccaggcaagc acgtggtacg agaagagcag 88200 tcattaggaa ggccatttgg aaaagcacatcctctctgtt cacgtgagat attttacatc 88260 ctcattcctc atcgcaagct tcctgggatttggagtgtca cagacaagag ggttggggga 88320 ggccagtagg tatggatttg tttatattaaaatgagcata tgaatattta tatgtttata 88380 ttaaaacata tatgtttgtt tatattacaatgagcatatg aatatttata tgtttatatt 88440 aaaacatata tgtttgttta tattaaaatgagcatatgaa tatttctgta tacttcagat 88500 aaacattctt ttccataaat aagcttcatcatccagaagc catgttgaaa gttggtaatc 88560 aaggatagga agtgtttcca agggttgtcagtgattaaat caaccttacc ttagcataca 88620 tgta 88624 2 4530 DNA Homosapiens 2 atggggaaga acaaactcct tcatccaagt ctggttcttc tcctcttggtcctcctgccc 60 acagacgcct cagtctctgg aaaaccgcag tatatggttc tggtcccctccctgctccac 120 actgagacca ctgagaaggg ctgtgtcctt ctgagctacc tgaatgagacagtgactgta 180 agtgcttcct tggagtctgt caggggaaac aggagcctct tcactgacctggaggcggag 240 aatgacgtac tccactgtgt cgccttcgct gtcccaaagt cttcatccaatgaggaggta 300 atgttcctca ctgtccaagt gaaaggacca acccaagaat ttaagaagcggaccacagtg 360 atggttaaga acgaggacag tctggtcttt gtccagacag acaaatcaatctacaaacca 420 gggcagacag tgaaatttcg tgttgtctcc atggatgaaa actttcaccccctgaatgag 480 ttgattccac tagtatacat tcaggatccc aaaggaaatc gcatcgcacaatggcagagt 540 ttccagttag agggtggcct caagcaattt tcttttcccc tctcatcagagcccttccag 600 ggctcctaca aggtggtggt acagaagaaa tcaggtggaa ggacagagcaccctttcacc 660 gtggaggaat ttgttcttcc caagtttgaa gtacaagtaa cagtgccaaagataatcacc 720 atcttggaag aagagatgaa tgtatcagtg tgtggcctat acacatatgggaagcctgtc 780 cctggacatg tgactgtgag catttgcaga aagtatagtg acgcttccgactgccacggt 840 gaagattcac aggctttctg tgagaaattc agtggacagc taaacagccatggctgcttc 900 tatcagcaag taaaaaccaa ggtcttccag ctgaagagga aggagtatgaaatgaaactt 960 cacactgagg cccagatcca agaagaagga acagtggtgg aattgactggaaggcagtcc 1020 agtgaaatca caagaaccat aaccaaactc tcatttgtga aagtggactcacactttcga 1080 cagggaattc ccttctttgg gcaggtgcgc ctagtagatg ggaaaggcgtccctatacca 1140 aataaagtca tattcatcag aggaaatgaa gcaaactatt actccaatgctaccacggat 1200 gagcatggcc ttgtacagtt ctctatcaac accaccaatg ttatgggtacctctcttact 1260 gttagggtca attacaagga tcgtagtccc tgttacggct accagtgggtgtcagaagaa 1320 cacgaagagg cacatcacac tgcttatctt gtgttctccc caagcaagagctttgtccac 1380 cttgagccca tgtctcatga actaccctgt ggccatactc agacagtccaggcacattat 1440 attctgaatg gaggcaccct gctggggctg aagaagctct ccttctattatctgataatg 1500 gcaaagggag gcattgtccg aactgggact catggactgc ttgtgaagcaggaagacatg 1560 aagggccatt tttccatctc aatccctgtg aagtcagaca ttgctcctgtcgctcggttg 1620 ctcatctatg ctgttttacc taccggggac gtgattgggg attctgcaaaatatgatgtt 1680 gaaaattgtc tggccaacaa ggtggatttg agcttcagcc catcacaaagtctcccagcc 1740 tcacacgccc acctgcgagt cacagcggct cctcagtccg tctgcgccctccgtgctgtg 1800 gaccaaagcg tgctgctcat gaagcctgat gctgagctct cggcgtcctcggtttacaac 1860 ctgctaccag aaaaggacct cactggcttc cctgggcctt tgaatgaccaggacaatgaa 1920 gactgcatca atcgtcataa tgtctatatt aatggaatca catatactccagtatcaagt 1980 acaaatgaaa aggatatgta cagcttccta gaggacatgg gcttaaaggcattcaccaac 2040 tcaaagattc gtaaacccaa aatgtgtcca cagcttcaac agtatgaaatgcatggacct 2100 gaaggtctac gtgtaggttt ttatgagtca gatgtaatgg gaagaggccatgcacgcctg 2160 gtgcatgttg aagagcctca cacggagacc gtacgaaagt acttccctgagacatggatc 2220 tgggatttgg tggtggtaaa ctcagcaggt gtggctgagg taggagtaacagtccctgac 2280 accatcaccg agtggaaggc aggggccttc tgcctgtctg aagatgctggacttggtatc 2340 tcttccactg cctctctccg agccttccag cccttctttg tggagctcacaatgccttac 2400 tctgtgattc gtggagaggc cttcacactc aaggccacgg tcctaaactaccttcccaaa 2460 tgcatccggg tcagtgtgca gctggaagcc tctcccgcct tcctagctgtcccagtggag 2520 aaggaacaag cgcctcactg catctgtgca aacgggcggc aaactgtgtcctgggcagta 2580 accccaaagt cattaggaaa tgtgaatttc actgtgagcg cagaggcactagagtctcaa 2640 gagctgtgtg ggactgaggt gccttcagtt cctgaacacg gaaggaaagacacagtcatc 2700 aagcctctgt tggttgaacc tgaaggacta gagaaggaaa caacattcaactccctactt 2760 tgtccatcag gtggtgaggt ttctgaagaa ttatccctga aactgccaccaaatgtggta 2820 gaagaatctg cccgagcttc tgtctcagtt ttgggagaca tattaggctctgccatgcaa 2880 aacacacaaa atcttctcca gatgccctat ggctgtggag agcagaatatggtcctcttt 2940 gctcctaaca tctatgtact ggattatcta aatgaaacac agcagcttactccagagatc 3000 aagtccaagg ccattggcta tctcaacact ggttaccaga gacagttgaactacaaacac 3060 tatgatggct cctacagcac ctttggggag cgatatggca ggaaccagggcaacacctgg 3120 ctcacagcct ttgttctgaa gacttttgcc caagctcgag cctacatcttcatcgatgaa 3180 gcacacatta cccaagccct catatggctc tcccagaggc agaaggacaatggctgtttc 3240 aggagctctg ggtcactgct caacaatgcc ataaagggag gagtagaagatgaagtgacc 3300 ctctccgcct atatcaccat cgcccttctg gagattcctc tcacagtcactcaccctgtt 3360 gtccgcaatg ccctgttttg cctggagtca gcctggaaga cagcacaagaaggggaccat 3420 ggcagccatg tatataccaa agcactgctg gcctatgctt ttgccctggcaggtaaccag 3480 gacaagagga aggaagtact caagtcactt aatgaggaag ctgtgaagaaagacaactct 3540 gtccattggg agcgccctca gaaacccaag gcaccagtgg ggcatttttacgaaccccag 3600 gctccctctg ctgaggtgga gatgacatcc tatgtgctcc tcgcttatctcacggcccag 3660 ccagccccaa cctcggagga cctgacctct gcaaccaaca tcgtgaagtggatcacgaag 3720 cagcagaatg cccagggcgg tttctcctcc acccaggaca cagtggtggctctccatgct 3780 ctgtccaaat atggagcagc cacatttacc aggactggga aggctgcacaggtgactatc 3840 cagtcttcag ggacattttc cagcaaattc caagtggaca acaacaaccgcctgttactg 3900 cagcaggtct cattgccaga gctgcctggg gaatacagca tgaaagtgacaggagaagga 3960 tgtgtctacc tccagacatc cttgaaatac aatattctcc cagaaaaggaagagttcccc 4020 tttgctttag gagtgcagac tctgcctcaa acttgtgatg aacccaaagcccacaccagc 4080 ttccaaatct ccctaagtgt cagttacaca gggagccgct ctgcctccaacatggcgatc 4140 gttgatgtga agatggtctc tggcttcatt cccctgaagc caacagtgaaaatgcttgaa 4200 agatctaacc atgtgagccg gacagaagtc agcagcaacc atgtcttgatttaccttgat 4260 aaggtgtcaa atcagacact gagcttgttc ttcacggttc tgcaagatgtcccagtaaga 4320 gatctgaaac cagccatagt gaaagtctat gattactacg agacgggtgatttgcaattg 4380 ctgagtacaa tgctccttgc agcaaagatc ttggaaatgc ttgaagaccacaaggctgaa 4440 aagtgctttg ctggagtcct gttctcagag ctccacagaa gacacgtgtttttgtatctt 4500 taaagacttg atgaataaac actttttctg 4530 3 4577 DNA Homosapiens 3 gctacaatcc atctggtctc ctccagctcc ttctttctgc aacatggggaagaacaaact 60 ccttcatcca agtctggttc ttctcctctt ggtcctcctg cccacagacgcctcagtctc 120 tggaaaaccg cagtatatgg ttctggtccc ctccctgctc cacactgagaccactgagaa 180 gggctgtgtc cttctgagct acctgaatga gacagtgact gtaagtgcttccttggagtc 240 tgtcagggga aacaggagcc tcttcactga cctggaggcg gagaatgacgtactccactg 300 tgtcgccttc gctgtcccaa agtcttcatc caatgaggag gtaatgttcctcactgtcca 360 agtgaaagga ccaacccaag aatttaagaa gcggaccaca gtgatggttaagaacgagga 420 cagtctggtc tttgtccaga cagacaaatc aatctacaaa ccagggcagacagtgaaatt 480 tcgtgttgtc tccatggatg aaaactttca ccccctgaat gagttgattccactagtata 540 cattcaggat cccaaaggaa atcgcatcgc acaatggcag agtttccagttagagggtgg 600 cctcaagcaa ttttcttttc ccctctcatc agagcccttc cagggctcctacaaggtggt 660 ggtacagaag aaatcaggtg gaaggacaga gcaccctttc accgtggaggaatttgttct 720 tcccaagttt gaagtacaag taacagtgcc aaagataatc accatcttggaagaagagat 780 gaatgtatca gtgtgtggcc tatacacata tgggaagcct gtccctggacatgtgactgt 840 gagcatttgc agaaagtata gtgacgcttc cgactgccac ggtgaagattcacaggcttt 900 ctgtgagaaa ttcagtggac agctaaacag ccatggctgc ttctatcagcaagtaaaaac 960 caaggtcttc cagctgaaga ggaaggagta tgaaatgaaa cttcacactgaggcccagat 1020 ccaagaagaa ggaacagtgg tggaattgac tggaaggcag tccagtgaaatcacaagaac 1080 cataaccaaa ctctcatttg tgaaagtgga ctcacacttt cgacagggaattcccttctt 1140 tgggcaggtg cgcctagtag atgggaaagg cgtccctata ccaaataaagtcatattcat 1200 cagaggaaat gaagcaaact attactccaa tgctaccacg gatgagcatggccttgtaca 1260 gttctctatc aacaccacca acgttatggg tacctctctt actgttagggtcaattacaa 1320 ggatcgtagt ccctgttacg gctaccagtg ggtgtcagaa gaacacgaagaggcacatca 1380 cactgcttat cttgtgttct ccccaagcaa gagctttgtc caccttgagcccatgtctca 1440 tgaactaccc tgtggccata ctcagacagt ccaggcacat tatattctgaatggaggcac 1500 cctgctgggg ctgaagaagc tctcctttta ttatctgata atggcaaagggaggcattgt 1560 ccgaactggg actcatggac tgcttgtgaa gcaggaagac atgaagggccatttttccat 1620 ctcaatccct gtgaagtcag acattgctcc tgtcgctcgg ttgctcatctatgctgtttt 1680 acctaccggg gacgtgattg gggattctgc aaaatatgat gttgaaaattgtctggccaa 1740 caaggtggat ttgagcttca gcccatcaca aagtctccca gcctcacacgcccacctgcg 1800 agtcacagcg gctcctcagt ccgtctgcgc cctccgtgct gtggaccaaagcgtgctgct 1860 catgaagcct gatgctgagc tctcggcgtc ctcggtttac aacctgctaccagaaaagga 1920 cctcactggc ttccctgggc ctttgaatga ccaggacgat gaagactgcatcaatcgtca 1980 taatgtctat attaatggaa tcacatatac tccagtatca agtacaaatgaaaaggatat 2040 gtacagcttc ctagaggaca tgggcttaaa ggcattcacc aactcaaagattcgtaaacc 2100 caaaatgtgt ccacagcttc aacagtatga aatgcatgga cctgaaggtctacgtgtagg 2160 tttttatgag tcagatgtaa tgggaagagg ccatgcacgc ctggtgcatgttgaagagcc 2220 tcacacggag accgtacgaa agtacttccc tgagacatgg atctgggatttggtggtggt 2280 aaactcagca ggggtggctg aggtaggagt aacagtccct gacaccatcaccgagtggaa 2340 ggcaggggcc ttctgcctgt ctgaagatgc tggacttggt atctcttccactgcctctct 2400 ccgagccttc cagcccttct ttgtggagct tacaatgcct tactctgtgattcgtggaga 2460 ggccttcaca ctcaaggcca cggtcctaaa ctaccttccc aaatgcatccgggtcagtgt 2520 gcagctggaa gcctctcccg ccttccttgc tgtcccagtg gagaaggaacaagcgcctca 2580 ctgcatctgt gcaaacgggc ggcaaactgt gtcctgggca gtaaccccaaagtcattagg 2640 aaatgtgaat ttcactgtga gcgcagaggc actagagtct caagagctgtgtgggactga 2700 ggtgccttca gttcctgaac acggaaggaa agacacagtc atcaagcctctgttggttga 2760 acctgaagga ctagagaagg aaacaacatt caactcccta ctttgtccatcaggtggtga 2820 ggtttctgaa gaattatccc tgaaactgcc accaaatgtg gtagaagaatctgcccgagc 2880 ttctgtctca gttttgggag acatattagg ctctgccatg caaaacacacaaaatcttct 2940 ccagatgccc tatggctgtg gagagcagaa tatggtcctc tttgctcctaacatctatgt 3000 actggattat ctaaatgaaa cacagcagct tactccagag gtcaagtccaaggccattgg 3060 ctatctcaac actggttacc agagacagtt gaactacaaa cactatgatggctcctacag 3120 cacctttggg gagcgatatg gcaggaacca gggcaacacc tggctcacagcctttgttct 3180 gaagactttt gcccaagctc gagcctacat cttcatcgat gaagcacacattacccaagc 3240 cctcatatgg ctctcccaga ggcagaagga caatggctgt ttcaggagctctgggtcact 3300 gctcaacaat gccataaagg gaggagtaga agatgaagtg accctctccgcctatatcac 3360 catcgccctt ctggagattc ctctcacagt cactcaccct gttgtccgcaatgccctgtt 3420 ttgcctggag tcagcctgga agacagcaca agaaggggac catggcagccatgtatatac 3480 caaagcactg ctggcctatg cttttgccct ggcaggtaac caggacaagaggaaggaagt 3540 actcaagtca cttaatgagg aagctgtgaa gaaagacaac tctgtccattgggagcgccc 3600 tcagaaaccc aaggcaccag tggggcattt ttacgaaccc caggctccctctgctgaggt 3660 ggagatgaca tcctatgtgc tcctcgctta tctcacggcc cagccagccccaacctcgga 3720 ggacctgacc tctgcaacca acatcgtgaa gtggatcacg aagcagcagaatgcccaggg 3780 cggtttctcc tccacccagg acacagtggt ggctctccat gctctgtccaaatatggagc 3840 cgccacattt accaggactg ggaaggctgc acaggtgact atccagtcttcagggacatt 3900 ttccagcaaa ttccaagtgg acaacaacaa tcgcctgtta ctgcagcaggtctcattgcc 3960 agagctgcct ggggaataca gcatgaaagt gacaggagaa ggatgtgtctacctccagac 4020 ctccttgaaa tacaatattc tcccagaaaa ggaagagttc ccctttgctttaggagtgca 4080 gactctgcct caaacttgtg atgaacccaa agcccacacc agcttccaaatctccctaag 4140 tgtcagttac acagggagcc gctctgcctc caacatggcg atcgttgatgtgaagatggt 4200 ctctggcttc attcccctga agccaacagt gaaaatgctt gaaagatctaaccatgtgag 4260 ccggacagaa gtcagcagca accatgtctt gatttacctt gataaggtgtcaaatcagac 4320 actgagcttg ttcttcacgg ttctgcaaga tgtcccagta agagatctcaaaccagccat 4380 agtgaaagtc tatgattact acgagacgga tgagtttgca atcgctgagtacaatgctcc 4440 ttgcagcaaa gatcttggaa atgcttgaag accacaaggc tgaaaagtgctttgctggag 4500 tcctgttctc tgagctccac agaagacacg tgtttttgta tctttaaagacttgatgaat 4560 aaacactttt tctggtc 4577 4 2041 DNA Homo sapiens 4cccgccttcc tagctgtccc agtggagaag gaacaagcgc ctcactgcat ctgtgcaaac 60gggcggcaaa ctgtgtcctg ggcagtaacc ccaaagtcat taggaaatgt gaatttcact 120gtgagcgcag aggcactaga gtctcaagag ctgtgtggga ctgaggtgcc ttcagttcct 180gaacacggaa ggaaagacac agtcatcaag cctctgttgg ttgaacctga aggactagag 240aaggaaacaa cattcaactc cctactttgt ccatcaggtg gtgaggtttc tgaagaatta 300tccctgaaac tgccaccaaa tgtggtagaa gaatctgccc gagcttctgt ctcagttttg 360ggagacatat taggctctgc catgcaaaac acacaaaatc ttctccagat gccctatggc 420tgtggagagc agaatatggt cctctttgct cctaacatct atgtactgga ttatctaaat 480gaaacacagc agcttactcc agagatcaag tccaaggcca ttggctatct caacactggt 540taccagagac agttgaacta caaacactat gatggctcct acagcacctt tggggagcga 600tatggcagga accagggcaa cacctggctc acagcctttg ttctgaagac ttttgcccaa 660gctcgagcct acatcttcat cgatgaagca cacattaccc aagccctcat atggctctcc 720cagaggcaga aggacaatgg ctgtttcagg agctctgggt cactgctcaa caatgccata 780aagggaggag tagaagatga agtgaccctc tccgcctata tcaccatcgc ccttctggag 840attcctctca cagtcactca ccctgttgtc cgcaatgccc tgttttgcct ggagtcagcc 900tggaagacag cacaagaagg ggaccatggc agccatgtat ataccaaaga cctgctggcc 960tatgcttttg ccctggcagg taaccaggac aagaggaagg aagtactcaa gtcacttaat 1020gaggaagctg tgaagaaaga caactctgtc cattgggagc gccctcagaa acccaaggca 1080ccagtggggg atttttacga accccaggct ccctctgctg aggtggagat gacatcctat 1140gtgctcctcg cttatctcac ggcccagcca gccccaacct cggaggacct gacctctgca 1200accaacatcg tgaagtggat cacgaagcag cagaatgccc agggcggttt ctcctccacc 1260caggacacag tggtggctct ccatgctctg tccaaatatg gagcagccac atttaccagg 1320actgggaagg ctgcacaggt gactatccag tcttcaggga cattttccag caaattccaa 1380gtggacaaca acaaccgcct gttactgcag caggtctcat tgccagagct gcctggggaa 1440tacagcatga aagtgacagg agaaggatgt gtctacctcc agacatcctt gaaatacaat 1500attctcccag aaaaggaaga gttccccttt gctttaggag tgcagactct gcctcaaact 1560tgtgatgaac ccaaagccca caccagcttc caaatctccc taagtgtcag ttacacaggg 1620agccgctctg cctccaacat ggcgatcgtt gatgtgaaga tggtctctgg cttcattccc 1680ctgaagccaa cagtgaaaat gcttgaaaga tctaaccatg tgagccggac agaagtcagc 1740agcaaccatg tcttgattta ccttgataag gtgtcaaatc agacactgag cttgttcttc 1800acggttctgc aagatgtccc agtaagagat ctgaaaccag ccatagtgaa agtctatgat 1860tactacgaga cggatgagtt tgcaattgct gagtacaatg ctccttgcag caaagatctt 1920ggaaatgctt gaagaccaca aggctgaaaa gtgctttgct ggagtcctgt tctcagagct 1980ccacagaaga cacgtgtttt tgtatcttta aagacttgat gaataaacac tttttctggt 2040 c2041 5 4577 DNA Homo sapiens 5 gctacaatcc atctggtctc ctccagctccttctttctgc aacatgggga agaacaaact 60 ccttcatcca agtctggttc ttctcctcttggtcctcctg cccacagacg cctcagtctc 120 tggaaaaccg cagtatatgg ttctggtcccctccctgctc cacactgaga ccactgagaa 180 gggctgtgtc cttctgagct acctgaatgagacagtgact gtaagtgctt ccttggagtc 240 tgtcagggga aacaggagcc tcttcactgacctggaggcg gagaatgacg tactccactg 300 tgtcgccttc gctgtcccaa agtcttcatccaatgaggag gtaatgttcc tcactgtcca 360 agtgaaagga ccaacccaag aatttaagaagcggaccaca gtgatggtta agaacgagga 420 cagtctggtc tttgtccaga cagacaaatcaatctacaaa ccagggcaga cagtgaaatt 480 tcgtgttgtc tccatggatg aaaactttcaccccctgaat gagttgattc cactagtata 540 cattcaggat cccaaaggaa atcgcatcgcacaatggcag agtttccagt tagagggtgg 600 cctcaagcaa ttttcttttc ccctctcatcagagcccttc cagggctcct acaaggtggt 660 ggtacagaag aaatcaggtg gaaggacagagcaccctttc accgtggagg aatttgttct 720 tcccaagttt gaagtacaag taacagtgccaaagataatc accatcttgg aagaagagat 780 gaatgtatca gtgtgtggcc tatacacatatgggaagcct gtccctggac atgtgactgt 840 gagcatttgc agaaagtata gtgacgcttccgactgccac ggtgaagatt cacaggcttt 900 ctgtgagaaa ttcagtggac agctaaacagccatggctgc ttctatcagc aagtaaaaac 960 caaggtcttc cagctgaaga ggaaggagtatgaaatgaaa cttcacactg aggcccagat 1020 ccaagaagaa ggaacagtgg tggaattgactggaaggcag tccagtgaaa tcacaagaac 1080 cataaccaaa ctctcatttg tgaaagtggactcacacttt cgacagggaa ttcccttctt 1140 tgggcaggtg cgcctagtag atgggaaaggcgtccctata ccaaataaag tcatattcat 1200 cagaggaaat gaagcaaact attactccaatgctaccacg gatgagcatg gccttgtaca 1260 gttctctatc aacaccacca acgttatgggtacctctctt actgttaggg tcaattacaa 1320 ggatcgtagt ccctgttacg gctaccagtgggtgtcagaa gaacacgaag aggcacatca 1380 cactgcttat cttgtgttct ccccaagcaagagctttgtc caccttgagc ccatgtctca 1440 tgaactaccc tgtggccata ctcagacagtccaggcacat tatattctga atggaggcac 1500 cctgctgggg ctgaagaagc tctccttttattatctgata atggcaaagg gaggcattgt 1560 ccgaactggg actcatggac tgcttgtgaagcaggaagac atgaagggcc atttttccat 1620 ctcaatccct gtgaagtcag acattgctcctgtcgctcgg ttgctcatct atgctgtttt 1680 acctaccggg gacgtgattg gggattctgcaaaatatgat gttgaaaatt gtctggccaa 1740 caaggtggat ttgagcttca gcccatcacaaagtctccca gcctcacacg cccacctgcg 1800 agtcacagcg gctcctcagt ccgtctgcgccctccgtgct gtggaccaaa gcgtgctgct 1860 catgaagcct gatgctgagc tctcggcgtcctcggtttac aacctgctac cagaaaagga 1920 cctcactggc ttccctgggc ctttgaatgaccaggacgat gaagactgca tcaatcgtca 1980 taatgtctat attaatggaa tcacatatactccagtatca agtacaaatg aaaaggatat 2040 gtacagcttc ctagaggaca tgggcttaaaggcattcacc aactcaaaga ttcgtaaacc 2100 caaaatgtgt ccacagcttc aacagtatgaaatgcatgga cctgaaggtc tacgtgtagg 2160 tttttatgag tcagatgtaa tgggaagaggccatgcacgc ctggtgcatg ttgaagagcc 2220 tcacacggag accgtacgaa agtacttccctgagacatgg atctgggatt tggtggtggt 2280 aaactcagca ggggtggctg aggtaggagtaacagtccct gacaccatca ccgagtggaa 2340 ggcaggggcc ttctgcctgt ctgaagatgctggacttggt atctcttcca ctgcctctct 2400 ccgagccttc cagcccttct ttgtggagcttacaatgcct tactctgtga ttcgtggaga 2460 ggccttcaca ctcaaggcca cggtcctaaactaccttccc aaatgcatcc gggtcagtgt 2520 gcagctggaa gcctctcccg ccttccttgctgtcccagtg gagaaggaac aagcgcctca 2580 ctgcatctgt gcaaacgggc ggcaaactgtgtcctgggca gtaaccccaa agtcattagg 2640 aaatgtgaat ttcactgtga gcgcagaggcactagagtct caagagctgt gtgggactga 2700 ggtgccttca gttcctgaac acggaaggaaagacacagtc atcaagcctc tgttggttga 2760 acctgaagga ctagagaagg aaacaacattcaactcccta ctttgtccat caggtggtga 2820 ggtttctgaa gaattatccc tgaaactgccaccaaatgtg gtagaagaat ctgcccgagc 2880 ttctgtctca gttttgggag acatattaggctctgccatg caaaacacac aaaatcttct 2940 ccagatgccc tatggctgtg gagagcagaatatggtcctc tttgctccta acatctatgt 3000 actggattat ctaaatgaaa cacagcagcttactccagag gtcaagtcca aggccattgg 3060 ctatctcaac actggttacc agagacagttgaactacaaa cactatgatg gctcctacag 3120 cacctttggg gagcgatatg gcaggaaccagggcaacacc tggctcacag cctttgttct 3180 gaagactttt gcccaagctc gagcctacatcttcatcgat gaagcacaca ttacccaagc 3240 cctcatatgg ctctcccaga ggcagaaggacaatggctgt ttcaggagct ctgggtcact 3300 gctcaacaat gccataaagg gaggagtagaagatgaagtg accctctccg cctatatcac 3360 catcgccctt ctggagattc ctctcacagtcactcaccct gttgtccgca atgccctgtt 3420 ttgcctggag tcagcctgga agacagcacaagaaggggac catggcagcc atgtatatac 3480 caaagcactg ctggcctatg cttttgccctggcaggtaac caggacaaga ggaaggaagt 3540 actcaagtca cttaatgagg aagctgtgaagaaagacaac tctgtccatt gggagcgccc 3600 tcagaaaccc aaggcaccag tggggcatttttacgaaccc caggctccct ctgctgaggt 3660 ggagatgaca tcctatgtgc tcctcgcttatctcacggcc cagccagccc caacctcgga 3720 ggacctgacc tctgcaacca acatcgtgaagtggatcacg aagcagcaga atgcccaggg 3780 cggtttctcc tccacccagg acacagtggtggctctccat gctctgtcca aatatggagc 3840 cgccacattt accaggactg ggaaggctgcacaggtgact atccagtctt cagggacatt 3900 ttccagcaaa ttccaagtgg acaacaacaatcgcctgtta ctgcagcagg tctcattgcc 3960 agagctgcct ggggaataca gcatgaaagtgacaggagaa ggatgtgtct acctccagac 4020 ctccttgaaa tacaatattc tcccagaaaaggaagagttc ccctttgctt taggagtgca 4080 gactctgcct caaacttgtg atgaacccaaagcccacacc agcttccaaa tctccctaag 4140 tgtcagttac acagggagcc gctctgcctccaacatggcg atcgttgatg tgaagatggt 4200 ctctggcttc attcccctga agccaacagtgaaaatgctt gaaagatcta accatgtgag 4260 ccggacagaa gtcagcagca accatgtcttgatttacctt gataaggtgt caaatcagac 4320 actgagcttg ttcttcacgg ttctgcaagatgtcccagta agagatctca aaccagccat 4380 agtgaaagtc tatgattact acgagacggatgagtttgca atcgctgagt acaatgctcc 4440 ttgcagcaaa gatcttggaa atgcttgaagaccacaaggc tgaaaagtgc tttgctggag 4500 tcctgttctc tgagctccac agaagacacgtgtttttgta tctttaaaga cttgatgaat 4560 aaacactttt tctggtc 4577 6 256 DNAHomo sapiens 6 tatattttat aatatatatt tactgattag atgataattt tctttgcaggacatgggctt 60 aaaggcattc accaactcaa agattcgtaa acccaaaatg tgtccacagcttcaacagta 120 tgaaatgcat ggacctgaag gtctacgtgt aggtttttat ggtaaacaaaaaattaataa 180 atatatattg cctaatatat tcaccaaatt ttaaattttt taaaagatacaatgtgacaa 240 aaattaacaa acaaaa 256 7 4576 DNA Homo sapiens 7tacaatacag tctgttctcc tccagctcct tctttctgca acatggggaa gaacaaactc 60cttcatccaa gtctggttct tctcctcttg gtcctcctgc ccacagacgc ctcagtctct 120ggaaaaccgc agtatatggt tctggtcccc tccctgctcc acactgagac cactgagaag 180ggctgtgtcc ttctgagcta cctgaatgag acagtgactg taagtgcttc cttggagtct 240gtcaggggaa acaggagcct cttcactgac ctggaggcgg agaatgacgt actccactgt 300gtcgccttcg ctgtcccaaa gtcttcatcc aatgaggagg taatgttcct cactgtccaa 360gtgaaaggac caacccaaga atttaagaag cggaccacag tgatggttaa gaacgaggac 420agtctggtct ttgtccagac agacaaatca atctacaaac cagggcagac agtgaaattt 480cgtgttgtct ccatggatga aaactttcac cccctgaatg agttgattcc actagtatac 540attcaggatc ccaaaggaaa tcgcatcgca caatggcaga gtttccagtt agagggtggc 600ctcaagcaat tttcttttcc cctctcatca gagcccttcc agggctccta caaggtggtg 660gtacagaaga aatcaggtgg aaggacagag caccctttca ccgtggagga atttgttctt 720cccaagtttg aagtacaagt aacagtgcca aagataatca ccatcttgga agaagagatg 780aatgtatcag tgtgtggcct atacacatat gggaagcctg tccctggaca tgtgactgtg 840agcatttgca gaaagtatag tgacgcttcc gactgccacg gtgaagattc acaggctttc 900tgtgagaaat tcagtggaca gctaaacagc catggctgct tctatcagca agtaaaaacc 960aaggtcttcc agctgaagag gaaggagtat gaaatgaaac ttcacactga ggcccagatc 1020caagaagaag gaacagtggt ggaattgact ggaaggcagt ccagtgaaat cacaagaacc 1080ataaccaaac tctcatttgt gaaagtggac tcacactttc gacagggaat tcccttcttt 1140gggcaggtgc gcctagtaga tgggaaaggc gtccctatac caaataaagt catattcatc 1200agaggaaatg aagcaaacta ttactccaat gctaccacgg atgagcatgg ccttgtacag 1260ttctctatca acaccaccaa tgttatgggt acctctctta ctgttagggt caattacaag 1320gatcgtagtc cctgttacgg ctaccagtgg gtgtcagaag aacacgaaga ggcacatcac 1380actgcttatc ttgtgttctc cccaagcaag agctttgtcc accttgagcc catgtctcat 1440gaactaccct gtggccatac tcagacagtc caggcacatt atattctgaa tggaggcacc 1500ctgctggggc tgaagaagct ctccttctat tatctgataa tggcaaaggg aggcattgtc 1560cgaactggga ctcatggact gcttgtgaag caggaagaca tgaagggcca tttttccatc 1620tcaatccctg tgaagtcaga cattgctcct gtcgctcggt tgctcatcta tgctgtttta 1680cctaccgggg acgtgattgg ggattctgca aaatatgatg ttgaaaattg tctggccaac 1740aaggtggatt tgagcttcag cccatcacaa agtctcccag cctcacacgc ccacctgcga 1800gtcacagcgg ctcctcagtc cgtctgcgcc ctccgtgctg tggaccaaag cgtgctgctc 1860atgaagcctg atgctgagct ctcggcgtcc tcggtttaca acctgctacc agaaaaggac 1920ctcactggct tccctgggcc tttgaatgac caggacaatg aagactgcat caatcgtcat 1980aatgtctata ttaatggaat cacatatact ccagtatcaa gtacaaatga aaaggatatg 2040tacagcttcc tagaggacat gggcttaaag gcattcacca actcaaagat tcgtaaaccc 2100aaaatgtgtc cacagcttca acagtatgaa atgcatggac ctgaaggtct acgtgtaggt 2160ttttatgagt cagatgtaat gggaagaggc catgcacgcc tggtgcatgt tgaagagcct 2220cacacggaga ccgtacgaaa gtacttccct gagacatgga tctgggattt ggtggtggta 2280aactcagcag gtgtggctga ggtaggagta acagtccctg acaccatcac cgagtggaag 2340gcaggggcct tctgcctgtc tgaagatgct ggacttggta tctcttccac tgcctctctc 2400cgagccttcc agcccttctt tgtggagctc acaatgcctt actctgtgat tcgtggagag 2460gccttcacac tcaaggccac ggtcctaaac taccttccca aatgcatccg ggtcagtgtg 2520cagctggaag cctctcccgc cttcctagct gtcccagtgg agaaggaaca agcgcctcac 2580tgcatctgtg caaacgggcg gcaaactgtg tcctgggcag taaccccaaa gtcattagga 2640aatgtgaatt tcactgtgag cgcagaggca ctagagtctc aagagctgtg tgggactgag 2700gtgccttcag ttcctgaaca cggaaggaaa gacacagtca tcaagcctct gttggttgaa 2760cctgaaggac tagagaagga aacaacattc aactccctac tttgtccatc aggtggtgag 2820gtttctgaag aattatccct gaaactgcca ccaaatgtgg tagaagaatc tgcccgagct 2880tctgtctcag ttttgggaga catattaggc tctgccatgc aaaacacaca aaatcttctc 2940cagatgccct atggctgtgg agagcagaat atggtcctct ttgctcctaa catctatgta 3000ctggattatc taaatgaaac acagcagctt actccagaga tcaagtccaa ggccattggc 3060tatctcaaca ctggttacca gagacagttg aactacaaac actatgatgg ctcctacagc 3120acctttgggg agcgatatgg caggaaccag ggcaacacct ggctcacagc ctttgttctg 3180aagacttttg cccaagctcg agcctacatc ttcatcgatg aagcacacat tacccaagcc 3240ctcatatggc tctcccagag gcagaaggac aatggctgtt tcaggagctc tgggtcactg 3300ctcaacaatg ccataaaggg aggagtagaa gatgaagtga ccctctccgc ctatatcacc 3360atcgcccttc tggagattcc tctcacagtc actcaccctg ttgtccgcaa tgccctgttt 3420tgcctggagt cagcctggaa gacagcacaa gaaggggacc atggcagcca tgtatatacc 3480aaagcactgc tggcctatgc ttttgccctg gcaggtaacc aggacaagag gaaggaagta 3540ctcaagtcac ttaatgagga agctgtgaag aaagacaact ctgtccattg ggagcgccct 3600cagaaaccca aggcaccagt ggggcatttt tacgaacccc aggctccctc tgctgaggtg 3660gagatgacat cctatgtgct cctcgcttat ctcacggccc agccagcccc aacctcggag 3720gacctgacct ctgcaaccaa catcgtgaag tggatcacga agcagcagaa tgcccagggc 3780ggtttctcct ccacccagga cacagtggtg gctctccatg ctctgtccaa atatggagca 3840gccacattta ccaggactgg gaaggctgca caggtgacta tccagtcttc agggacattt 3900tccagcaaat tccaagtgga caacaacaac cgcctgttac tgcagcaggt ctcattgcca 3960gagctgcctg gggaatacag catgaaagtg acaggagaag gatgtgtcta cctccagaca 4020tccttgaaat acaatattct cccagaaaag gaagagttcc cctttgcttt aggagtgcag 4080actctgcctc aaacttgtga tgaacccaaa gcccacacca gcttccaaat ctccctaagt 4140gtcagttaca cagggagccg ctctgcctcc aacatggcga tcgttgatgt gaagatggtc 4200tctggcttca ttcccctgaa gccaacagtg aaaatgcttg aaagatctaa ccatgtgagc 4260cggacagaag tcagcagcaa ccatgtcttg atttaccttg ataaggtgtc aaatcagaca 4320ctgagcttgt tcttcacggt tctgcaagat gtcccagtaa gagatctgaa accagccata 4380gtgaaagtct atgattacta cgagacggat gagtttgcaa ttgctgagta caatgctcct 4440tgcagcaaag atcttggaaa tgcttgaaga ccacaaggct gaaaagtgct ttgctggagt 4500cctgttctca gagctccaca gaagacacgt gtttttgtat ctttaaagac ttgatgaata 4560aacacttttt ctggtc 4576 8 6487 DNA Homo sapiens 8 gaattctatt gtttgtagtaaattgtttta gtccaaacac taattcctct gtagcaaaca 60 taggatctaa taaaatggattatgtgtgga aatcagtcct ctttagaaac ctaaaggacc 120 aagtgtatcc tgattaaaaagataaaacgc tttctttctt tctttttgtt tttgtttttt 180 tgtttgtttg tttcgagacagaggctcgct ctgttgccag gctggagtgc agtggcgtga 240 tctcggctca ctgcaacctctgcctcccgg gtttaagcga ttctcgtgca tcagtctccc 300 gtgcagctgg gactacaggcgcacgcacca cacccagcta atttttgtag tttaagtaga 360 gacggggttt caccatgttggccaggatgg tctcaatctc ttgacctcat gatccacctg 420 cctcagtctc ccaaagtgctttttgataat tttgagaaat gatggaagca tattagaatg 480 aaaacaacct gaggatgtgcttttatcttt gtatattcaa atattttttc tcattaaaaa 540 gcagaaagtc cgggtatgatggttcatgcc tgtaacccta acactttgcg gggccgagat 600 aggaagatcc cgtgaggtcaggactttgag gctagcctga gcaacatggt aggaccctgt 660 ctccataaaa agcttaagaaaaaaattagc ggggcgtggt ggagtgcacc tgtagtctta 720 gctatttggg aggctgagatgggaggatca cttgagccta ggagttcaag gctgcactga 780 gctatgatct aaccactgtactccagcctg ggcaacagag caagaccctg tctctgaaaa 840 aaaaaaatac acacacacacacacacacac acacacacac acacacacat gttagtggga 900 tagcacaaat gagaaaaactctgctctttg atcactgagt acatctctgt agatatatat 960 ttccttcact gcagattttgcccaagatac ttcgtcaaag acaaagccag tacaccctct 1020 aatagggtga atatggttatgccacctact gagcttgttt ttgatactag ttaatatgta 1080 accagatgaa attgtcattatcgtcactgt caggactatg ggaagcttaa gtgttctctt 1140 ttcaaggaca atgtgcgctaactgtacaat tggtacaatt aaataagtta tattcagttc 1200 ctgggaagca ctatagcaatacaaggagaa aatttgattc tatttatttt tgttaaggcc 1260 cacctacctc ctaatcctaatttctctcat ttcccaaata ttccttgttt gttcttactg 1320 ttatgtgttt tcctgtattttgctcttcta ctttcttttc catggactat ctttttccct 1380 tccttttttt cgctctacccctttacctca gctttctagc agtatttgct aaatacttca 1440 aaactgtata gaactggttcaaattgtgtg ctcccttttc tgtcaagaac ttgctactca 1500 ggtaacccaa ttggtgatttttcctggaaa cactgatgga tgctgttcct atagcgaaac 1560 ccagaacaga gatgaaatagatgtcatcct cagccattag cattcaaact ataaaaatta 1620 atttacactg gtatagtaaggatcagaatg tcaaagctgt gttacaccta gcatcttgta 1680 tgaaactacc ccattaaggtgagaccacag atattattgc cccactattg gcatgaaagc 1740 tgaggctcag agcagttaactgagttaccc aggaccacac agctaagtta gaagtagggc 1800 tcaggtgtcc tggcaactaactggtccagt tattttttct ctcaagctcg ttttccctct 1860 cctaaagaat aggaggctctgtcgtggtga aaggcgattt tagtaatact ttccttttta 1920 tctgtgatta taatgaatgcggcatctctc ccattaagga tcattcctcc acccacattc 1980 ttaatacatc tgctgcatgcatccttcaga gacctccctc tgggatcatc ccttctcact 2040 ccaaaaagct caacttctcccctgtcattt gtacctccca ctcagcattt ttagaagcaa 2100 tatttcattc aaacttattcaagtttattt ccacctaaag aaatattcct ttcaccctgg 2160 catctccgtc aggtactgctctgttgtttt tctccccttc agacaaactg ccaaactggc 2220 tctagttcct cacattccccatcaccctca gcaagcttct gccccacacc ggcactgaaa 2280 cagctgaatc ccaatgtccttgtccttaaa cccagcagaa aaaaaaaatc aatcaattat 2340 ttgatttcac agcggcacttgacatgggta gccaggaatt tatcaatgac aacctttaca 2400 gatcatcttt gtaatttatcatgaggcatc aaatgaatgc tattaacatt aatccctcct 2460 attttaagtc attaatccaagtaaatgctc acttatttct agcatcttag aaaccattta 2520 aattatgtta cattatgaatcaatacatta taaaattata ccatcatttg taataatttt 2580 ttaaaatgtt gtgtgctattaacattgatg ccttggtata aagtcatgat cattctggtc 2640 tagtagcaat cttctattgactattctctt actaaagcgg tcccttccgt gggactcaga 2700 gacctcacac tctcctgcctgtgtttcttc ctctctaatt ggcccttctt gctccacttg 2760 ggtgctcctg cccattgcctagacaagagc attccctgta actctgtctt gggctctttt 2820 tctcttttca tcaacatcttctacgtgggt attatcatcc atttccatgg catcagcttg 2880 cccaataaac tgataaatccatagtctcta taagtacagc agatctcatc aagctagtgg 2940 cattcagact gctttaactttaaccaaaaa taagggattt tgtacatgtt caataagcag 3000 ttcccactgt gacactgtaatcacattttc acaattgtga cctaggacac ttagagtaaa 3060 ggatacagat gattgagacagaaatagtga caaagaaaaa taaggttagg atatagattt 3120 taatgctgta acagacctcaaaatacaatg gcttaactaa gagaatgcat ttctctgtca 3180 cataaaggtc ccaactggcgtagacttttg atgactcaag ggctcaggct gtgcctggtt 3240 tgtggttctg ccttccttaacacatggctt ccatctgatg agctacagca gtacctatca 3300 ctagtcagca tgtccacattccagcctggg caaggaagaa aggggaagcg cagaactgta 3360 cccttccttt tttaagtcatgaactgaaag ttgcatgtat cacttccact tgccctccag 3420 tcaccagaac ttagtcatatgccataccca gcttcaaggg agtgggttaa aaacatagaa 3480 gtcaactagg cagtctgcacccagcaaagg atcgggagtt ctattattaa agcagaattg 3540 gagaagtggt aacaggaaacaaccaccagc ctctgctgca tgtatatgaa acagatgttt 3600 cccaaatcac tattctcacttattctgtct gatacactgt attttttatt atattctctt 3660 tcatttttta aaatcctggtcatgactcac agggcatgat gttacaaccc acttagatgc 3720 taacaccata atctgaaaaatattacctat attatgtcta atattggcca cttgaagtat 3780 ggctagccta aattgatctatgttgtaagt ataaaattca caccagcttg tgaaaacaaa 3840 ttatgaaaaa aaagtctttaagatatcatt aacaatttta tattggctaa atgttgaaat 3900 gatcatattt tggatatattggattaaata aaatacacta ttaaaattaa tttaatgttt 3960 ctctttatgt ggttactagaaaatttaaaa tttaaaatta cacagggcga tcacattcta 4020 tttctagtag accacactgctgtaagctca agattcaaat gtcaaactcc tgtgaatatt 4080 aatacgtgaa tatcccacaagcacttactc catcttccca accctcagcc cttctgtcct 4140 ccttctgctc ccaccaatctgtgtttcttc tgtttcactc acccagctaa aggcaacaca 4200 attcactccg tgacgagccaggaaaatgga aagacacatt ttcctttatt cctcacattg 4260 atatattcac tgagcactataattacctct taaatatgat ataaatctgc aagctctttt 4320 caataccacc acaaattccatagttcaaaa tgccatcagc tttcacctat attattacac 4380 cagctcccat ctggtcttcctgcatcctgg atcacctctt tctagctgcc ctttcaaatt 4440 tcaataagag caagctttccaggaaacaaa cctgaagtca atccactgag tactcctctg 4500 aataccttaa tattgttgacaaattccttt ctgatttgaa gtatcagaaa ggaatatttc 4560 ctccatacca aatagttttcatttcatgca tgtgccgtga ttcttctccc tcctttgcat 4620 ctgtcattcg ttatgcttagaaagctcttt tcatctcttt gttcttcgag acaaccacta 4680 ctcatacttc agagcttaatttacattttg ctttccctca aaattttttt aaaaggttcc 4740 aggtctgggt tatgtgctctcttatgtgct cccagagcat cctgaacttc tgcaataata 4800 tgtttggcta ctgtattttatacagtagtt ttatattgta ttttatacag taggtgttat 4860 attgtatttt atacagtagttgtttttctg tctgtttttg ccccaacaag aatgtaaaat 4920 ctttaagtgc ctgttttcatacttatttga ccaccctatc tctagaatct tgcatgatgt 4980 ctagccctag taggatcaaaaaatacttac aaagcaactg aatagctaca tgaatagatg 5040 gatgaataaa tgcatgggtggatggatgga ttaatgaaat catttatatg acttaaagtt 5100 tgcagaggag tatcatatttggaaggcagt aaggaagtct gtgtagtcga tggtaaaggc 5160 aattgggaag tttgttaggcacaataggtc aaaatttgtt tttgaagtcc tgttacttca 5220 cgtttctttg tttcactttcttaaaacagg aaactctttt ctatgatcat tcttccaggg 5280 cctggctctt catctgcaacccagtaatat ccctaatgtc aaaaagctac tggtttaatt 5340 cgtgccattt tcaaagaggactactgaatt ctgatgtggc ttcaaacatt taggttaggc 5400 atatctaatg gagaacttgcagccacactg acttgtagtg aaatatctat tttgagcctg 5460 cccagtgttg cttaaattgtagttttcctt gccagctatt catacaagag atgtgagaag 5520 caccataaaa ggcgttgtgaggagttgtgg gggagtgagg gagagaagag gttgaaaagc 5580 ttattagctg ctgtacggtaaaagtgagct cttacgggaa tgggaatgta gttttagccc 5640 tccagggatt ctatttagcccgccaggaat taaccttgac tataaatagg ccatcaatga 5700 cctttccaga gaatgttcagagacctcaac tttgtttaga gatcttgtgt gggtggaact 5760 tcctgtttgc acacagagcagcataaagcc cagttgcttt gggaagtgtt tgggaccaga 5820 tggattgtag ggagtagggtacaatacagt ctggtctcct ccagctcctt ctttctgcaa 5880 catggggaag aacaaactccttcatccaag tctggttctt ctcctcttgg tcctcctgcc 5940 cacagacgcc tcagtctctggaaaaccgtg agttccacac agagagcgtg aagcatgaac 6000 ctagagtcct tcatttattgcagatttttc tttatatcat tcctttttct ttcctatgat 6060 actgtcatct tcttatctctaagattcctt ccagatttta caaatctagt ttactcatta 6120 cttgcttact tttaatcattcttccccaac tctctgaagc tctaatatgc aaagccttcc 6180 taaggggtgt cagaaatttttagcttttta aaagaataaa ttttagatat tcacattcat 6240 attgatctac ttgagaccatgctatttatc ttttcttatt tcctctttct caagggtcca 6300 ttttctattt tataaaaataaagacaattc tctcccacaa ccaaacatgg aacaatgccc 6360 tggagtataa aaatctatagagtgccaaat aaaggaacaa tttgaaatac tggtgttgat 6420 attgaaaaag caagggactctaatgtcaga agagaaatcc ttttgcagat gaggtggtga 6480 tgaattc 6487 9 1500 PRTHomo sapiens 9 Met Gly Lys Asn Lys Leu Leu His Pro Ser Leu Val Leu LeuLeu Leu 1 5 10 15 Val Leu Leu Pro Thr Asp Ala Ser Val Ser Gly Lys ProGln Tyr Met 20 25 30 Val Leu Val Pro Ser Leu Leu His Thr Glu Thr Thr GluLys Gly Cys 35 40 45 Val Leu Leu Ser Tyr Leu Asn Glu Thr Val Thr Val SerAla Ser Leu 50 55 60 Glu Ser Val Arg Gly Asn Arg Ser Leu Phe Thr Asp LeuGlu Ala Glu 65 70 75 80 Asn Asp Val Leu His Cys Val Ala Phe Ala Val ProLys Ser Ser Ser 85 90 95 Asn Glu Glu Val Met Phe Leu Thr Val Gln Val LysGly Pro Thr Gln 100 105 110 Glu Phe Lys Lys Arg Thr Thr Val Met Val LysAsn Glu Asp Ser Leu 115 120 125 Val Phe Val Gln Thr Asp Lys Ser Ile TyrLys Pro Gly Gln Thr Val 130 135 140 Lys Phe Arg Val Val Ser Met Asp GluAsn Phe His Pro Leu Asn Glu 145 150 155 160 Leu Ile Pro Leu Val Tyr IleGln Asp Pro Lys Gly Asn Arg Ile Ala 165 170 175 Gln Trp Gln Ser Phe GlnLeu Glu Gly Gly Leu Lys Gln Phe Ser Phe 180 185 190 Pro Leu Ser Ser GluPro Phe Gln Gly Ser Tyr Lys Val Val Val Gln 195 200 205 Lys Lys Ser GlyGly Arg Thr Glu His Pro Phe Thr Val Glu Glu Phe 210 215 220 Val Leu ProLys Phe Glu Val Gln Val Thr Val Pro Lys Ile Ile Thr 225 230 235 240 IleLeu Glu Glu Glu Met Asn Val Ser Val Cys Gly Leu Tyr Thr Tyr 245 250 255Gly Lys Pro Val Pro Gly His Val Thr Val Ser Ile Cys Arg Lys Tyr 260 265270 Ser Asp Ala Ser Asp Cys His Gly Glu Asp Ser Gln Ala Phe Cys Glu 275280 285 Lys Phe Ser Gly Gln Leu Asn Ser His Gly Cys Phe Tyr Gln Gln Val290 295 300 Lys Thr Lys Val Phe Gln Leu Lys Arg Lys Glu Tyr Glu Met LysLeu 305 310 315 320 His Thr Glu Ala Gln Ile Gln Glu Glu Gly Thr Val ValGlu Leu Thr 325 330 335 Gly Arg Gln Ser Ser Glu Ile Thr Arg Thr Ile ThrLys Leu Ser Phe 340 345 350 Val Lys Val Asp Ser His Phe Arg Gln Gly IlePro Phe Phe Gly Gln 355 360 365 Val Arg Leu Val Asp Gly Lys Gly Val ProIle Pro Asn Lys Val Ile 370 375 380 Phe Ile Arg Gly Asn Glu Ala Asn TyrTyr Ser Asn Ala Thr Thr Asp 385 390 395 400 Glu His Gly Leu Val Gln PheSer Ile Asn Thr Thr Asn Val Met Gly 405 410 415 Thr Ser Leu Thr Val ArgVal Asn Tyr Lys Asp Arg Ser Pro Cys Tyr 420 425 430 Gly Tyr Gln Trp ValSer Glu Glu His Glu Glu Ala His His Thr Ala 435 440 445 Tyr Leu Val PheSer Pro Ser Lys Ser Phe Val His Leu Glu Pro Met 450 455 460 Ser His GluLeu Pro Cys Gly His Thr Gln Thr Val Gln Ala His Tyr 465 470 475 480 IleLeu Asn Gly Gly Thr Leu Leu Gly Leu Lys Lys Leu Ser Phe Tyr 485 490 495Tyr Leu Ile Met Ala Lys Gly Gly Ile Val Arg Thr Gly Thr His Gly 500 505510 Leu Leu Val Lys Gln Glu Asp Met Lys Gly His Phe Ser Ile Ser Ile 515520 525 Pro Val Lys Ser Asp Ile Ala Pro Val Ala Arg Leu Leu Ile Tyr Ala530 535 540 Val Leu Pro Thr Gly Asp Val Ile Gly Asp Ser Ala Lys Tyr AspVal 545 550 555 560 Glu Asn Cys Leu Ala Asn Lys Val Asp Leu Ser Phe SerPro Ser Gln 565 570 575 Ser Leu Pro Ala Ser His Ala His Leu Arg Val ThrAla Ala Pro Gln 580 585 590 Ser Val Cys Ala Leu Arg Ala Val Asp Gln SerVal Leu Leu Met Lys 595 600 605 Pro Asp Ala Glu Leu Ser Ala Ser Ser ValTyr Asn Leu Leu Pro Glu 610 615 620 Lys Asp Leu Thr Gly Phe Pro Gly ProLeu Asn Asp Gln Asp Asn Glu 625 630 635 640 Asp Cys Ile Asn Arg His AsnVal Tyr Ile Asn Gly Ile Thr Tyr Thr 645 650 655 Pro Val Ser Ser Thr AsnGlu Lys Asp Met Tyr Ser Phe Leu Glu Asp 660 665 670 Met Gly Leu Lys AlaPhe Thr Asn Ser Lys Ile Arg Lys Pro Lys Met 675 680 685 Cys Pro Gln LeuGln Gln Tyr Glu Met His Gly Pro Glu Gly Leu Arg 690 695 700 Val Gly PheTyr Glu Ser Asp Val Met Gly Arg Gly His Ala Arg Leu 705 710 715 720 ValHis Val Glu Glu Pro His Thr Glu Thr Val Arg Lys Tyr Phe Pro 725 730 735Glu Thr Trp Ile Trp Asp Leu Val Val Val Asn Ser Ala Gly Val Ala 740 745750 Glu Val Gly Val Thr Val Pro Asp Thr Ile Thr Glu Trp Lys Ala Gly 755760 765 Ala Phe Cys Leu Ser Glu Asp Ala Gly Leu Gly Ile Ser Ser Thr Ala770 775 780 Ser Leu Arg Ala Phe Gln Pro Phe Phe Val Glu Leu Thr Met ProTyr 785 790 795 800 Ser Val Ile Arg Gly Glu Ala Phe Thr Leu Lys Ala ThrVal Leu Asn 805 810 815 Tyr Leu Pro Lys Cys Ile Arg Val Ser Val Gln LeuGlu Ala Ser Pro 820 825 830 Ala Phe Leu Ala Val Pro Val Glu Lys Glu GlnAla Pro His Cys Ile 835 840 845 Cys Ala Asn Gly Arg Gln Thr Val Ser TrpAla Val Thr Pro Lys Ser 850 855 860 Leu Gly Asn Val Asn Phe Thr Val SerAla Glu Ala Leu Glu Ser Gln 865 870 875 880 Glu Leu Cys Gly Thr Glu ValPro Ser Val Pro Glu His Gly Arg Lys 885 890 895 Asp Thr Val Ile Lys ProLeu Leu Val Glu Pro Glu Gly Leu Glu Lys 900 905 910 Glu Thr Thr Phe AsnSer Leu Leu Cys Pro Ser Gly Gly Glu Val Ser 915 920 925 Glu Glu Leu SerLeu Lys Leu Pro Pro Asn Val Val Glu Glu Ser Ala 930 935 940 Arg Ala SerVal Ser Val Leu Gly Asp Ile Leu Gly Ser Ala Met Gln 945 950 955 960 AsnThr Gln Asn Leu Leu Gln Met Pro Tyr Gly Cys Gly Glu Gln Asn 965 970 975Met Val Leu Phe Ala Pro Asn Ile Tyr Val Leu Asp Tyr Leu Asn Glu 980 985990 Thr Gln Gln Leu Thr Pro Glu Ile Lys Ser Lys Ala Ile Gly Tyr Leu 9951000 1005 Asn Thr Gly Tyr Gln Arg Gln Leu Asn Tyr Lys His Tyr Asp GlySer 1010 1015 1020 Tyr Ser Thr Phe Gly Glu Arg Tyr Gly Arg Asn Gln GlyAsn Thr Trp 1025 1030 1035 1040 Leu Thr Ala Phe Val Leu Lys Thr Phe AlaGln Ala Arg Ala Tyr Ile 1045 1050 1055 Phe Ile Asp Glu Ala His Ile ThrGln Ala Leu Ile Trp Leu Ser Gln 1060 1065 1070 Arg Gln Lys Asp Asn GlyCys Phe Arg Ser Ser Gly Ser Leu Leu Asn 1075 1080 1085 Asn Ala Ile LysGly Gly Val Glu Asp Glu Val Thr Leu Ser Ala Tyr 1090 1095 1100 Ile ThrIle Ala Leu Leu Glu Ile Pro Leu Thr Val Thr His Pro Val 1105 1110 11151120 Val Arg Asn Ala Leu Phe Cys Leu Glu Ser Ala Trp Lys Thr Ala Gln1125 1130 1135 Glu Gly Asp His Gly Ser His Val Tyr Thr Lys Ala Leu LeuAla Tyr 1140 1145 1150 Ala Phe Ala Leu Ala Gly Asn Gln Asp Lys Arg LysGlu Val Leu Lys 1155 1160 1165 Ser Leu Asn Glu Glu Ala Val Lys Lys AspAsn Ser Val His Trp Glu 1170 1175 1180 Arg Pro Gln Lys Pro Lys Ala ProVal Gly His Phe Tyr Glu Pro Gln 1185 1190 1195 1200 Ala Pro Ser Ala GluVal Glu Met Thr Ser Tyr Val Leu Leu Ala Tyr 1205 1210 1215 Leu Thr AlaGln Pro Ala Pro Thr Ser Glu Asp Leu Thr Ser Ala Thr 1220 1225 1230 AsnIle Val Lys Trp Ile Thr Lys Gln Gln Asn Ala Gln Gly Gly Phe 1235 12401245 Ser Ser Thr Gln Asp Thr Val Val Ala Leu His Ala Leu Ser Lys Tyr1250 1255 1260 Gly Ala Ala Thr Phe Thr Arg Thr Gly Lys Ala Ala Gln ValThr Ile 1265 1270 1275 1280 Gln Ser Ser Gly Thr Phe Ser Ser Lys Phe GlnVal Asp Asn Asn Asn 1285 1290 1295 Arg Leu Leu Leu Gln Gln Val Ser LeuPro Glu Leu Pro Gly Glu Tyr 1300 1305 1310 Ser Met Lys Val Thr Gly GluGly Cys Val Tyr Leu Gln Thr Ser Leu 1315 1320 1325 Lys Tyr Asn Ile LeuPro Glu Lys Glu Glu Phe Pro Phe Ala Leu Gly 1330 1335 1340 Val Gln ThrLeu Pro Gln Thr Cys Asp Glu Pro Lys Ala His Thr Ser 1345 1350 1355 1360Phe Gln Ile Ser Leu Ser Val Ser Tyr Thr Gly Ser Arg Ser Ala Ser 13651370 1375 Asn Met Ala Ile Val Asp Val Lys Met Val Ser Gly Phe Ile ProLeu 1380 1385 1390 Lys Pro Thr Val Lys Met Leu Glu Arg Ser Asn His ValSer Arg Thr 1395 1400 1405 Glu Val Ser Ser Asn His Val Leu Ile Tyr LeuAsp Lys Val Ser Asn 1410 1415 1420 Gln Thr Leu Ser Leu Phe Phe Thr ValLeu Gln Asp Val Pro Val Arg 1425 1430 1435 1440 Asp Leu Lys Pro Ala IleVal Lys Val Tyr Asp Tyr Tyr Glu Thr Gly 1445 1450 1455 Asp Leu Gln LeuLeu Ser Thr Met Leu Leu Ala Ala Lys Ile Leu Glu 1460 1465 1470 Met LeuGlu Asp His Lys Ala Glu Lys Cys Phe Ala Gly Val Leu Phe 1475 1480 1485Ser Glu Leu His Arg Arg His Val Phe Leu Tyr Leu 1490 1495 1500 10 1474PRT Homo sapiens 10 Met Gly Lys Asn Lys Leu Leu His Pro Ser Leu Val LeuLeu Leu Leu 1 5 10 15 Val Leu Leu Pro Thr Asp Ala Ser Val Ser Gly LysPro Gln Tyr Met 20 25 30 Val Leu Val Pro Ser Leu Leu His Thr Glu Thr ThrGlu Lys Gly Cys 35 40 45 Val Leu Leu Ser Tyr Leu Asn Glu Thr Val Thr ValSer Ala Ser Leu 50 55 60 Glu Ser Val Arg Gly Asn Arg Ser Leu Phe Thr AspLeu Glu Ala Glu 65 70 75 80 Asn Asp Val Leu His Cys Val Ala Phe Ala ValPro Lys Ser Ser Ser 85 90 95 Asn Glu Glu Val Met Phe Leu Thr Val Gln ValLys Gly Pro Thr Gln 100 105 110 Glu Phe Lys Lys Arg Thr Thr Val Met ValLys Asn Glu Asp Ser Leu 115 120 125 Val Phe Val Gln Thr Asp Lys Ser IleTyr Lys Pro Gly Gln Thr Val 130 135 140 Lys Phe Arg Val Val Ser Met AspGlu Asn Phe His Pro Leu Asn Glu 145 150 155 160 Leu Ile Pro Leu Val TyrIle Gln Asp Pro Lys Gly Asn Arg Ile Ala 165 170 175 Gln Trp Gln Ser PheGln Leu Glu Gly Gly Leu Lys Gln Phe Ser Phe 180 185 190 Pro Leu Ser SerGlu Pro Phe Gln Gly Ser Tyr Lys Val Val Val Gln 195 200 205 Lys Lys SerGly Gly Arg Thr Glu His Pro Phe Thr Val Glu Glu Phe 210 215 220 Val LeuPro Lys Phe Glu Val Gln Val Thr Val Pro Lys Ile Ile Thr 225 230 235 240Ile Leu Glu Glu Glu Met Asn Val Ser Val Cys Gly Leu Tyr Thr Tyr 245 250255 Gly Lys Pro Val Pro Gly His Val Thr Val Ser Ile Cys Arg Lys Tyr 260265 270 Ser Asp Ala Ser Asp Cys His Gly Glu Asp Ser Gln Ala Phe Cys Glu275 280 285 Lys Phe Ser Gly Gln Leu Asn Ser His Gly Cys Phe Tyr Gln GlnVal 290 295 300 Lys Thr Lys Val Phe Gln Leu Lys Arg Lys Glu Tyr Glu MetLys Leu 305 310 315 320 His Thr Glu Ala Gln Ile Gln Glu Glu Gly Thr ValVal Glu Leu Thr 325 330 335 Gly Arg Gln Ser Ser Glu Ile Thr Arg Thr IleThr Lys Leu Ser Phe 340 345 350 Val Lys Val Asp Ser His Phe Arg Gln GlyIle Pro Phe Phe Gly Gln 355 360 365 Val Arg Leu Val Asp Gly Lys Gly ValPro Ile Pro Asn Lys Val Ile 370 375 380 Phe Ile Arg Gly Asn Glu Ala AsnTyr Tyr Ser Asn Ala Thr Thr Asp 385 390 395 400 Glu His Gly Leu Val GlnPhe Ser Ile Asn Thr Thr Asn Val Met Gly 405 410 415 Thr Ser Leu Thr ValArg Val Asn Tyr Lys Asp Arg Ser Pro Cys Tyr 420 425 430 Gly Tyr Gln TrpVal Ser Glu Glu His Glu Glu Ala His His Thr Ala 435 440 445 Tyr Leu ValPhe Ser Pro Ser Lys Ser Phe Val His Leu Glu Pro Met 450 455 460 Ser HisGlu Leu Pro Cys Gly His Thr Gln Thr Val Gln Ala His Tyr 465 470 475 480Ile Leu Asn Gly Gly Thr Leu Leu Gly Leu Lys Lys Leu Ser Phe Tyr 485 490495 Tyr Leu Ile Met Ala Lys Gly Gly Ile Val Arg Thr Gly Thr His Gly 500505 510 Leu Leu Val Lys Gln Glu Asp Met Lys Gly His Phe Ser Ile Ser Ile515 520 525 Pro Val Lys Ser Asp Ile Ala Pro Val Ala Arg Leu Leu Ile TyrAla 530 535 540 Val Leu Pro Thr Gly Asp Val Ile Gly Asp Ser Ala Lys TyrAsp Val 545 550 555 560 Glu Asn Cys Leu Ala Asn Lys Val Asp Leu Ser PheSer Pro Ser Gln 565 570 575 Ser Leu Pro Ala Ser His Ala His Leu Arg ValThr Ala Ala Pro Gln 580 585 590 Ser Val Cys Ala Leu Arg Ala Val Asp GlnSer Val Leu Leu Met Lys 595 600 605 Pro Asp Ala Glu Leu Ser Ala Ser SerVal Tyr Asn Leu Leu Pro Glu 610 615 620 Lys Asp Leu Thr Gly Phe Pro GlyPro Leu Asn Asp Gln Asp Asp Glu 625 630 635 640 Asp Cys Ile Asn Arg HisAsn Val Tyr Ile Asn Gly Ile Thr Tyr Thr 645 650 655 Pro Val Ser Ser ThrAsn Glu Lys Asp Met Tyr Ser Phe Leu Glu Asp 660 665 670 Met Gly Leu LysAla Phe Thr Asn Ser Lys Ile Arg Lys Pro Lys Met 675 680 685 Cys Pro GlnLeu Gln Gln Tyr Glu Met His Gly Pro Glu Gly Leu Arg 690 695 700 Val GlyPhe Tyr Glu Ser Asp Val Met Gly Arg Gly His Ala Arg Leu 705 710 715 720Val His Val Glu Glu Pro His Thr Glu Thr Val Arg Lys Tyr Phe Pro 725 730735 Glu Thr Trp Ile Trp Asp Leu Val Val Val Asn Ser Ala Gly Val Ala 740745 750 Glu Val Gly Val Thr Val Pro Asp Thr Ile Thr Glu Trp Lys Ala Gly755 760 765 Ala Phe Cys Leu Ser Glu Asp Ala Gly Leu Gly Ile Ser Ser ThrAla 770 775 780 Ser Leu Arg Ala Phe Gln Pro Phe Phe Val Glu Leu Thr MetPro Tyr 785 790 795 800 Ser Val Ile Arg Gly Glu Ala Phe Thr Leu Lys AlaThr Val Leu Asn 805 810 815 Tyr Leu Pro Lys Cys Ile Arg Val Ser Val GlnLeu Glu Ala Ser Pro 820 825 830 Ala Phe Leu Ala Val Pro Val Glu Lys GluGln Ala Pro His Cys Ile 835 840 845 Cys Ala Asn Gly Arg Gln Thr Val SerTrp Ala Val Thr Pro Lys Ser 850 855 860 Leu Gly Asn Val Asn Phe Thr ValSer Ala Glu Ala Leu Glu Ser Gln 865 870 875 880 Glu Leu Cys Gly Thr GluVal Pro Ser Val Pro Glu His Gly Arg Lys 885 890 895 Asp Thr Val Ile LysPro Leu Leu Val Glu Pro Glu Gly Leu Glu Lys 900 905 910 Glu Thr Thr PheAsn Ser Leu Leu Cys Pro Ser Gly Gly Glu Val Ser 915 920 925 Glu Glu LeuSer Leu Lys Leu Pro Pro Asn Val Val Glu Glu Ser Ala 930 935 940 Arg AlaSer Val Ser Val Leu Gly Asp Ile Leu Gly Ser Ala Met Gln 945 950 955 960Asn Thr Gln Asn Leu Leu Gln Met Pro Tyr Gly Cys Gly Glu Gln Asn 965 970975 Met Val Leu Phe Ala Pro Asn Ile Tyr Val Leu Asp Tyr Leu Asn Glu 980985 990 Thr Gln Gln Leu Thr Pro Glu Val Lys Ser Lys Ala Ile Gly Tyr Leu995 1000 1005 Asn Thr Gly Tyr Gln Arg Gln Leu Asn Tyr Lys His Tyr AspGly Ser 1010 1015 1020 Tyr Ser Thr Phe Gly Glu Arg Tyr Gly Arg Asn GlnGly Asn Thr Trp 1025 1030 1035 1040 Leu Thr Ala Phe Val Leu Lys Thr PheAla Gln Ala Arg Ala Tyr Ile 1045 1050 1055 Phe Ile Asp Glu Ala His IleThr Gln Ala Leu Ile Trp Leu Ser Gln 1060 1065 1070 Arg Gln Lys Asp AsnGly Cys Phe Arg Ser Ser Gly Ser Leu Leu Asn 1075 1080 1085 Asn Ala IleLys Gly Gly Val Glu Asp Glu Val Thr Leu Ser Ala Tyr 1090 1095 1100 IleThr Ile Ala Leu Leu Glu Ile Pro Leu Thr Val Thr His Pro Val 1105 11101115 1120 Val Arg Asn Ala Leu Phe Cys Leu Glu Ser Ala Trp Lys Thr AlaGln 1125 1130 1135 Glu Gly Asp His Gly Ser His Val Tyr Thr Lys Ala LeuLeu Ala Tyr 1140 1145 1150 Ala Phe Ala Leu Ala Gly Asn Gln Asp Lys ArgLys Glu Val Leu Lys 1155 1160 1165 Ser Leu Asn Glu Glu Ala Val Lys LysAsp Asn Ser Val His Trp Glu 1170 1175 1180 Arg Pro Gln Lys Pro Lys AlaPro Val Gly His Phe Tyr Glu Pro Gln 1185 1190 1195 1200 Ala Pro Ser AlaGlu Val Glu Met Thr Ser Tyr Val Leu Leu Ala Tyr 1205 1210 1215 Leu ThrAla Gln Pro Ala Pro Thr Ser Glu Asp Leu Thr Ser Ala Thr 1220 1225 1230Asn Ile Val Lys Trp Ile Thr Lys Gln Gln Asn Ala Gln Gly Gly Phe 12351240 1245 Ser Ser Thr Gln Asp Thr Val Val Ala Leu His Ala Leu Ser LysTyr 1250 1255 1260 Gly Ala Ala Thr Phe Thr Arg Thr Gly Lys Ala Ala GlnVal Thr Ile 1265 1270 1275 1280 Gln Ser Ser Gly Thr Phe Ser Ser Lys PheGln Val Asp Asn Asn Asn 1285 1290 1295 Arg Leu Leu Leu Gln Gln Val SerLeu Pro Glu Leu Pro Gly Glu Tyr 1300 1305 1310 Ser Met Lys Val Thr GlyGlu Gly Cys Val Tyr Leu Gln Thr Ser Leu 1315 1320 1325 Lys Tyr Asn IleLeu Pro Glu Lys Glu Glu Phe Pro Phe Ala Leu Gly 1330 1335 1340 Val GlnThr Leu Pro Gln Thr Cys Asp Glu Pro Lys Ala His Thr Ser 1345 1350 13551360 Phe Gln Ile Ser Leu Ser Val Ser Tyr Thr Gly Ser Arg Ser Ala Ser1365 1370 1375 Asn Met Ala Ile Val Asp Val Lys Met Val Ser Gly Phe IlePro Leu 1380 1385 1390 Lys Pro Thr Val Lys Met Leu Glu Arg Ser Asn HisVal Ser Arg Thr 1395 1400 1405 Glu Val Ser Ser Asn His Val Leu Ile TyrLeu Asp Lys Val Ser Asn 1410 1415 1420 Gln Thr Leu Ser Leu Phe Phe ThrVal Leu Gln Asp Val Pro Val Arg 1425 1430 1435 1440 Asp Leu Lys Pro AlaIle Val Lys Val Tyr Asp Tyr Tyr Glu Thr Asp 1445 1450 1455 Glu Phe AlaIle Ala Glu Tyr Asn Ala Pro Cys Ser Lys Asp Leu Gly 1460 1465 1470 AsnAla 11 643 PRT Homo sapiens 11 Pro Ala Phe Leu Ala Val Pro Val Glu LysGlu Gln Ala Pro His Cys 1 5 10 15 Ile Cys Ala Asn Gly Arg Gln Thr ValSer Trp Ala Val Thr Pro Lys 20 25 30 Ser Leu Gly Asn Val Asn Phe Thr ValSer Ala Glu Ala Leu Glu Ser 35 40 45 Gln Glu Leu Cys Gly Thr Glu Val ProSer Val Pro Glu His Gly Arg 50 55 60 Lys Asp Thr Val Ile Lys Pro Leu LeuVal Glu Pro Glu Gly Leu Glu 65 70 75 80 Lys Glu Thr Thr Phe Asn Ser LeuLeu Cys Pro Ser Gly Gly Glu Val 85 90 95 Ser Glu Glu Leu Ser Leu Lys LeuPro Pro Asn Val Val Glu Glu Ser 100 105 110 Ala Arg Ala Ser Val Ser ValLeu Gly Asp Ile Leu Gly Ser Ala Met 115 120 125 Gln Asn Thr Gln Asn LeuLeu Gln Met Pro Tyr Gly Cys Gly Glu Gln 130 135 140 Asn Met Val Leu PheAla Pro Asn Ile Tyr Val Leu Asp Tyr Leu Asn 145 150 155 160 Glu Thr GlnGln Leu Thr Pro Glu Ile Lys Ser Lys Ala Ile Gly Tyr 165 170 175 Leu AsnThr Gly Tyr Gln Arg Gln Leu Asn Tyr Lys His Tyr Asp Gly 180 185 190 SerTyr Ser Thr Phe Gly Glu Arg Tyr Gly Arg Asn Gln Gly Asn Thr 195 200 205Trp Leu Thr Ala Phe Val Leu Lys Thr Phe Ala Gln Ala Arg Ala Tyr 210 215220 Ile Phe Ile Asp Glu Ala His Ile Thr Gln Ala Leu Ile Trp Leu Ser 225230 235 240 Gln Arg Gln Lys Asp Asn Gly Cys Phe Arg Ser Ser Gly Ser LeuLeu 245 250 255 Asn Asn Ala Ile Lys Gly Gly Val Glu Asp Glu Val Thr LeuSer Ala 260 265 270 Tyr Ile Thr Ile Ala Leu Leu Glu Ile Pro Leu Thr ValThr His Pro 275 280 285 Val Val Arg Asn Ala Leu Phe Cys Leu Glu Ser AlaTrp Lys Thr Ala 290 295 300 Gln Glu Gly Asp His Gly Ser His Val Tyr ThrLys Asp Leu Leu Ala 305 310 315 320 Tyr Ala Phe Ala Leu Ala Gly Asn GlnAsp Lys Arg Lys Glu Val Leu 325 330 335 Lys Ser Leu Asn Glu Glu Ala ValLys Lys Asp Asn Ser Val His Trp 340 345 350 Glu Arg Pro Gln Lys Pro LysAla Pro Val Gly Asp Phe Tyr Glu Pro 355 360 365 Gln Ala Pro Ser Ala GluVal Glu Met Thr Ser Tyr Val Leu Leu Ala 370 375 380 Tyr Leu Thr Ala GlnPro Ala Pro Thr Ser Glu Asp Leu Thr Ser Ala 385 390 395 400 Thr Asn IleVal Lys Trp Ile Thr Lys Gln Gln Asn Ala Gln Gly Gly 405 410 415 Phe SerSer Thr Gln Asp Thr Val Val Ala Leu His Ala Leu Ser Lys 420 425 430 TyrGly Ala Ala Thr Phe Thr Arg Thr Gly Lys Ala Ala Gln Val Thr 435 440 445Ile Gln Ser Ser Gly Thr Phe Ser Ser Lys Phe Gln Val Asp Asn Asn 450 455460 Asn Arg Leu Leu Leu Gln Gln Val Ser Leu Pro Glu Leu Pro Gly Glu 465470 475 480 Tyr Ser Met Lys Val Thr Gly Glu Gly Cys Val Tyr Leu Gln ThrSer 485 490 495 Leu Lys Tyr Asn Ile Leu Pro Glu Lys Glu Glu Phe Pro PheAla Leu 500 505 510 Gly Val Gln Thr Leu Pro Gln Thr Cys Asp Glu Pro LysAla His Thr 515 520 525 Ser Phe Gln Ile Ser Leu Ser Val Ser Tyr Thr GlySer Arg Ser Ala 530 535 540 Ser Asn Met Ala Ile Val Asp Val Lys Met ValSer Gly Phe Ile Pro 545 550 555 560 Leu Lys Pro Thr Val Lys Met Leu GluArg Ser Asn His Val Ser Arg 565 570 575 Thr Glu Val Ser Ser Asn His ValLeu Ile Tyr Leu Asp Lys Val Ser 580 585 590 Asn Gln Thr Leu Ser Leu PhePhe Thr Val Leu Gln Asp Val Pro Val 595 600 605 Arg Asp Leu Lys Pro AlaIle Val Lys Val Tyr Asp Tyr Tyr Glu Thr 610 615 620 Asp Glu Phe Ala IleAla Glu Tyr Asn Ala Pro Cys Ser Lys Asp Leu 625 630 635 640 Gly Asn Ala12 1474 PRT Homo sapiens 12 Met Gly Lys Asn Lys Leu Leu His Pro Ser LeuVal Leu Leu Leu Leu 1 5 10 15 Val Leu Leu Pro Thr Asp Ala Ser Val SerGly Lys Pro Gln Tyr Met 20 25 30 Val Leu Val Pro Ser Leu Leu His Thr GluThr Thr Glu Lys Gly Cys 35 40 45 Val Leu Leu Ser Tyr Leu Asn Glu Thr ValThr Val Ser Ala Ser Leu 50 55 60 Glu Ser Val Arg Gly Asn Arg Ser Leu PheThr Asp Leu Glu Ala Glu 65 70 75 80 Asn Asp Val Leu His Cys Val Ala PheAla Val Pro Lys Ser Ser Ser 85 90 95 Asn Glu Glu Val Met Phe Leu Thr ValGln Val Lys Gly Pro Thr Gln 100 105 110 Glu Phe Lys Lys Arg Thr Thr ValMet Val Lys Asn Glu Asp Ser Leu 115 120 125 Val Phe Val Gln Thr Asp LysSer Ile Tyr Lys Pro Gly Gln Thr Val 130 135 140 Lys Phe Arg Val Val SerMet Asp Glu Asn Phe His Pro Leu Asn Glu 145 150 155 160 Leu Ile Pro LeuVal Tyr Ile Gln Asp Pro Lys Gly Asn Arg Ile Ala 165 170 175 Gln Trp GlnSer Phe Gln Leu Glu Gly Gly Leu Lys Gln Phe Ser Phe 180 185 190 Pro LeuSer Ser Glu Pro Phe Gln Gly Ser Tyr Lys Val Val Val Gln 195 200 205 LysLys Ser Gly Gly Arg Thr Glu His Pro Phe Thr Val Glu Glu Phe 210 215 220Val Leu Pro Lys Phe Glu Val Gln Val Thr Val Pro Lys Ile Ile Thr 225 230235 240 Ile Leu Glu Glu Glu Met Asn Val Ser Val Cys Gly Leu Tyr Thr Tyr245 250 255 Gly Lys Pro Val Pro Gly His Val Thr Val Ser Ile Cys Arg LysTyr 260 265 270 Ser Asp Ala Ser Asp Cys His Gly Glu Asp Ser Gln Ala PheCys Glu 275 280 285 Lys Phe Ser Gly Gln Leu Asn Ser His Gly Cys Phe TyrGln Gln Val 290 295 300 Lys Thr Lys Val Phe Gln Leu Lys Arg Lys Glu TyrGlu Met Lys Leu 305 310 315 320 His Thr Glu Ala Gln Ile Gln Glu Glu GlyThr Val Val Glu Leu Thr 325 330 335 Gly Arg Gln Ser Ser Glu Ile Thr ArgThr Ile Thr Lys Leu Ser Phe 340 345 350 Val Lys Val Asp Ser His Phe ArgGln Gly Ile Pro Phe Phe Gly Gln 355 360 365 Val Arg Leu Val Asp Gly LysGly Val Pro Ile Pro Asn Lys Val Ile 370 375 380 Phe Ile Arg Gly Asn GluAla Asn Tyr Tyr Ser Asn Ala Thr Thr Asp 385 390 395 400 Glu His Gly LeuVal Gln Phe Ser Ile Asn Thr Thr Asn Val Met Gly 405 410 415 Thr Ser LeuThr Val Arg Val Asn Tyr Lys Asp Arg Ser Pro Cys Tyr 420 425 430 Gly TyrGln Trp Val Ser Glu Glu His Glu Glu Ala His His Thr Ala 435 440 445 TyrLeu Val Phe Ser Pro Ser Lys Ser Phe Val His Leu Glu Pro Met 450 455 460Ser His Glu Leu Pro Cys Gly His Thr Gln Thr Val Gln Ala His Tyr 465 470475 480 Ile Leu Asn Gly Gly Thr Leu Leu Gly Leu Lys Lys Leu Ser Phe Tyr485 490 495 Tyr Leu Ile Met Ala Lys Gly Gly Ile Val Arg Thr Gly Thr HisGly 500 505 510 Leu Leu Val Lys Gln Glu Asp Met Lys Gly His Phe Ser IleSer Ile 515 520 525 Pro Val Lys Ser Asp Ile Ala Pro Val Ala Arg Leu LeuIle Tyr Ala 530 535 540 Val Leu Pro Thr Gly Asp Val Ile Gly Asp Ser AlaLys Tyr Asp Val 545 550 555 560 Glu Asn Cys Leu Ala Asn Lys Val Asp LeuSer Phe Ser Pro Ser Gln 565 570 575 Ser Leu Pro Ala Ser His Ala His LeuArg Val Thr Ala Ala Pro Gln 580 585 590 Ser Val Cys Ala Leu Arg Ala ValAsp Gln Ser Val Leu Leu Met Lys 595 600 605 Pro Asp Ala Glu Leu Ser AlaSer Ser Val Tyr Asn Leu Leu Pro Glu 610 615 620 Lys Asp Leu Thr Gly PhePro Gly Pro Leu Asn Asp Gln Asp Asp Glu 625 630 635 640 Asp Cys Ile AsnArg His Asn Val Tyr Ile Asn Gly Ile Thr Tyr Thr 645 650 655 Pro Val SerSer Thr Asn Glu Lys Asp Met Tyr Ser Phe Leu Glu Asp 660 665 670 Met GlyLeu Lys Ala Phe Thr Asn Ser Lys Ile Arg Lys Pro Lys Met 675 680 685 CysPro Gln Leu Gln Gln Tyr Glu Met His Gly Pro Glu Gly Leu Arg 690 695 700Val Gly Phe Tyr Glu Ser Asp Val Met Gly Arg Gly His Ala Arg Leu 705 710715 720 Val His Val Glu Glu Pro His Thr Glu Thr Val Arg Lys Tyr Phe Pro725 730 735 Glu Thr Trp Ile Trp Asp Leu Val Val Val Asn Ser Ala Gly ValAla 740 745 750 Glu Val Gly Val Thr Val Pro Asp Thr Ile Thr Glu Trp LysAla Gly 755 760 765 Ala Phe Cys Leu Ser Glu Asp Ala Gly Leu Gly Ile SerSer Thr Ala 770 775 780 Ser Leu Arg Ala Phe Gln Pro Phe Phe Val Glu LeuThr Met Pro Tyr 785 790 795 800 Ser Val Ile Arg Gly Glu Ala Phe Thr LeuLys Ala Thr Val Leu Asn 805 810 815 Tyr Leu Pro Lys Cys Ile Arg Val SerVal Gln Leu Glu Ala Ser Pro 820 825 830 Ala Phe Leu Ala Val Pro Val GluLys Glu Gln Ala Pro His Cys Ile 835 840 845 Cys Ala Asn Gly Arg Gln ThrVal Ser Trp Ala Val Thr Pro Lys Ser 850 855 860 Leu Gly Asn Val Asn PheThr Val Ser Ala Glu Ala Leu Glu Ser Gln 865 870 875 880 Glu Leu Cys GlyThr Glu Val Pro Ser Val Pro Glu His Gly Arg Lys 885 890 895 Asp Thr ValIle Lys Pro Leu Leu Val Glu Pro Glu Gly Leu Glu Lys 900 905 910 Glu ThrThr Phe Asn Ser Leu Leu Cys Pro Ser Gly Gly Glu Val Ser 915 920 925 GluGlu Leu Ser Leu Lys Leu Pro Pro Asn Val Val Glu Glu Ser Ala 930 935 940Arg Ala Ser Val Ser Val Leu Gly Asp Ile Leu Gly Ser Ala Met Gln 945 950955 960 Asn Thr Gln Asn Leu Leu Gln Met Pro Tyr Gly Cys Gly Glu Gln Asn965 970 975 Met Val Leu Phe Ala Pro Asn Ile Tyr Val Leu Asp Tyr Leu AsnGlu 980 985 990 Thr Gln Gln Leu Thr Pro Glu Val Lys Ser Lys Ala Ile GlyTyr Leu 995 1000 1005 Asn Thr Gly Tyr Gln Arg Gln Leu Asn Tyr Lys HisTyr Asp Gly Ser 1010 1015 1020 Tyr Ser Thr Phe Gly Glu Arg Tyr Gly ArgAsn Gln Gly Asn Thr Trp 1025 1030 1035 1040 Leu Thr Ala Phe Val Leu LysThr Phe Ala Gln Ala Arg Ala Tyr Ile 1045 1050 1055 Phe Ile Asp Glu AlaHis Ile Thr Gln Ala Leu Ile Trp Leu Ser Gln 1060 1065 1070 Arg Gln LysAsp Asn Gly Cys Phe Arg Ser Ser Gly Ser Leu Leu Asn 1075 1080 1085 AsnAla Ile Lys Gly Gly Val Glu Asp Glu Val Thr Leu Ser Ala Tyr 1090 10951100 Ile Thr Ile Ala Leu Leu Glu Ile Pro Leu Thr Val Thr His Pro Val1105 1110 1115 1120 Val Arg Asn Ala Leu Phe Cys Leu Glu Ser Ala Trp LysThr Ala Gln 1125 1130 1135 Glu Gly Asp His Gly Ser His Val Tyr Thr LysAla Leu Leu Ala Tyr 1140 1145 1150 Ala Phe Ala Leu Ala Gly Asn Gln AspLys Arg Lys Glu Val Leu Lys 1155 1160 1165 Ser Leu Asn Glu Glu Ala ValLys Lys Asp Asn Ser Val His Trp Glu 1170 1175 1180 Arg Pro Gln Lys ProLys Ala Pro Val Gly His Phe Tyr Glu Pro Gln 1185 1190 1195 1200 Ala ProSer Ala Glu Val Glu Met Thr Ser Tyr Val Leu Leu Ala Tyr 1205 1210 1215Leu Thr Ala Gln Pro Ala Pro Thr Ser Glu Asp Leu Thr Ser Ala Thr 12201225 1230 Asn Ile Val Lys Trp Ile Thr Lys Gln Gln Asn Ala Gln Gly GlyPhe 1235 1240 1245 Ser Ser Thr Gln Asp Thr Val Val Ala Leu His Ala LeuSer Lys Tyr 1250 1255 1260 Gly Ala Ala Thr Phe Thr Arg Thr Gly Lys AlaAla Gln Val Thr Ile 1265 1270 1275 1280 Gln Ser Ser Gly Thr Phe Ser SerLys Phe Gln Val Asp Asn Asn Asn 1285 1290 1295 Arg Leu Leu Leu Gln GlnVal Ser Leu Pro Glu Leu Pro Gly Glu Tyr 1300 1305 1310 Ser Met Lys ValThr Gly Glu Gly Cys Val Tyr Leu Gln Thr Ser Leu 1315 1320 1325 Lys TyrAsn Ile Leu Pro Glu Lys Glu Glu Phe Pro Phe Ala Leu Gly 1330 1335 1340Val Gln Thr Leu Pro Gln Thr Cys Asp Glu Pro Lys Ala His Thr Ser 13451350 1355 1360 Phe Gln Ile Ser Leu Ser Val Ser Tyr Thr Gly Ser Arg SerAla Ser 1365 1370 1375 Asn Met Ala Ile Val Asp Val Lys Met Val Ser GlyPhe Ile Pro Leu 1380 1385 1390 Lys Pro Thr Val Lys Met Leu Glu Arg SerAsn His Val Ser Arg Thr 1395 1400 1405 Glu Val Ser Ser Asn His Val LeuIle Tyr Leu Asp Lys Val Ser Asn 1410 1415 1420 Gln Thr Leu Ser Leu PhePhe Thr Val Leu Gln Asp Val Pro Val Arg 1425 1430 1435 1440 Asp Leu LysPro Ala Ile Val Lys Val Tyr Asp Tyr Tyr Glu Thr Asp 1445 1450 1455 GluPhe Ala Ile Ala Glu Tyr Asn Ala Pro Cys Ser Lys Asp Leu Gly 1460 14651470 Asn Ala 13 1474 PRT Homo sapiens 13 Met Gly Lys Asn Lys Leu Leu HisPro Ser Leu Val Leu Leu Leu Leu 1 5 10 15 Val Leu Leu Pro Thr Asp AlaSer Val Ser Gly Lys Pro Gln Tyr Met 20 25 30 Val Leu Val Pro Ser Leu LeuHis Thr Glu Thr Thr Glu Lys Gly Cys 35 40 45 Val Leu Leu Ser Tyr Leu AsnGlu Thr Val Thr Val Ser Ala Ser Leu 50 55 60 Glu Ser Val Arg Gly Asn ArgSer Leu Phe Thr Asp Leu Glu Ala Glu 65 70 75 80 Asn Asp Val Leu His CysVal Ala Phe Ala Val Pro Lys Ser Ser Ser 85 90 95 Asn Glu Glu Val Met PheLeu Thr Val Gln Val Lys Gly Pro Thr Gln 100 105 110 Glu Phe Lys Lys ArgThr Thr Val Met Val Lys Asn Glu Asp Ser Leu 115 120 125 Val Phe Val GlnThr Asp Lys Ser Ile Tyr Lys Pro Gly Gln Thr Val 130 135 140 Lys Phe ArgVal Val Ser Met Asp Glu Asn Phe His Pro Leu Asn Glu 145 150 155 160 LeuIle Pro Leu Val Tyr Ile Gln Asp Pro Lys Gly Asn Arg Ile Ala 165 170 175Gln Trp Gln Ser Phe Gln Leu Glu Gly Gly Leu Lys Gln Phe Ser Phe 180 185190 Pro Leu Ser Ser Glu Pro Phe Gln Gly Ser Tyr Lys Val Val Val Gln 195200 205 Lys Lys Ser Gly Gly Arg Thr Glu His Pro Phe Thr Val Glu Glu Phe210 215 220 Val Leu Pro Lys Phe Glu Val Gln Val Thr Val Pro Lys Ile IleThr 225 230 235 240 Ile Leu Glu Glu Glu Met Asn Val Ser Val Cys Gly LeuTyr Thr Tyr 245 250 255 Gly Lys Pro Val Pro Gly His Val Thr Val Ser IleCys Arg Lys Tyr 260 265 270 Ser Asp Ala Ser Asp Cys His Gly Glu Asp SerGln Ala Phe Cys Glu 275 280 285 Lys Phe Ser Gly Gln Leu Asn Ser His GlyCys Phe Tyr Gln Gln Val 290 295 300 Lys Thr Lys Val Phe Gln Leu Lys ArgLys Glu Tyr Glu Met Lys Leu 305 310 315 320 His Thr Glu Ala Gln Ile GlnGlu Glu Gly Thr Val Val Glu Leu Thr 325 330 335 Gly Arg Gln Ser Ser GluIle Thr Arg Thr Ile Thr Lys Leu Ser Phe 340 345 350 Val Lys Val Asp SerHis Phe Arg Gln Gly Ile Pro Phe Phe Gly Gln 355 360 365 Val Arg Leu ValAsp Gly Lys Gly Val Pro Ile Pro Asn Lys Val Ile 370 375 380 Phe Ile ArgGly Asn Glu Ala Asn Tyr Tyr Ser Asn Ala Thr Thr Asp 385 390 395 400 GluHis Gly Leu Val Gln Phe Ser Ile Asn Thr Thr Asn Val Met Gly 405 410 415Thr Ser Leu Thr Val Arg Val Asn Tyr Lys Asp Arg Ser Pro Cys Tyr 420 425430 Gly Tyr Gln Trp Val Ser Glu Glu His Glu Glu Ala His His Thr Ala 435440 445 Tyr Leu Val Phe Ser Pro Ser Lys Ser Phe Val His Leu Glu Pro Met450 455 460 Ser His Glu Leu Pro Cys Gly His Thr Gln Thr Val Gln Ala HisTyr 465 470 475 480 Ile Leu Asn Gly Gly Thr Leu Leu Gly Leu Lys Lys LeuSer Phe Tyr 485 490 495 Tyr Leu Ile Met Ala Lys Gly Gly Ile Val Arg ThrGly Thr His Gly 500 505 510 Leu Leu Val Lys Gln Glu Asp Met Lys Gly HisPhe Ser Ile Ser Ile 515 520 525 Pro Val Lys Ser Asp Ile Ala Pro Val AlaArg Leu Leu Ile Tyr Ala 530 535 540 Val Leu Pro Thr Gly Asp Val Ile GlyAsp Ser Ala Lys Tyr Asp Val 545 550 555 560 Glu Asn Cys Leu Ala Asn LysVal Asp Leu Ser Phe Ser Pro Ser Gln 565 570 575 Ser Leu Pro Ala Ser HisAla His Leu Arg Val Thr Ala Ala Pro Gln 580 585 590 Ser Val Cys Ala LeuArg Ala Val Asp Gln Ser Val Leu Leu Met Lys 595 600 605 Pro Asp Ala GluLeu Ser Ala Ser Ser Val Tyr Asn Leu Leu Pro Glu 610 615 620 Lys Asp LeuThr Gly Phe Pro Gly Pro Leu Asn Asp Gln Asp Asp Glu 625 630 635 640 AspCys Ile Asn Arg His Asn Val Tyr Ile Asn Gly Ile Thr Tyr Thr 645 650 655Pro Val Ser Ser Thr Asn Glu Lys Asp Met Tyr Ser Phe Leu Glu Asp 660 665670 Met Gly Leu Lys Ala Phe Thr Asn Ser Lys Ile Arg Lys Pro Lys Met 675680 685 Cys Pro Gln Leu Gln Gln Tyr Glu Met His Gly Pro Glu Gly Leu Arg690 695 700 Val Gly Phe Tyr Glu Ser Asp Val Met Gly Arg Gly His Ala ArgLeu 705 710 715 720 Val His Val Glu Glu Pro His Thr Glu Thr Val Arg LysTyr Phe Pro 725 730 735 Glu Thr Trp Ile Trp Asp Leu Val Val Val Asn SerAla Gly Val Ala 740 745 750 Glu Val Gly Val Thr Val Pro Asp Thr Ile ThrGlu Trp Lys Ala Gly 755 760 765 Ala Phe Cys Leu Ser Glu Asp Ala Gly LeuGly Ile Ser Ser Thr Ala 770 775 780 Ser Leu Arg Ala Phe Gln Pro Phe PheVal Glu Leu Thr Met Pro Tyr 785 790 795 800 Ser Val Ile Arg Gly Glu AlaPhe Thr Leu Lys Ala Thr Val Leu Asn 805 810 815 Tyr Leu Pro Lys Cys IleArg Val Ser Val Gln Leu Glu Ala Ser Pro 820 825 830 Ala Phe Leu Ala ValPro Val Glu Lys Glu Gln Ala Pro His Cys Ile 835 840 845 Cys Ala Asn GlyArg Gln Thr Val Ser Trp Ala Val Thr Pro Lys Ser 850 855 860 Leu Gly AsnVal Asn Phe Thr Val Ser Ala Glu Ala Leu Glu Ser Gln 865 870 875 880 GluLeu Cys Gly Thr Glu Val Pro Ser Val Pro Glu His Gly Arg Lys 885 890 895Asp Thr Val Ile Lys Pro Leu Leu Val Glu Pro Glu Gly Leu Glu Lys 900 905910 Glu Thr Thr Phe Asn Ser Leu Leu Cys Pro Ser Gly Gly Glu Val Ser 915920 925 Glu Glu Leu Ser Leu Lys Leu Pro Pro Asn Val Val Glu Glu Ser Ala930 935 940 Arg Ala Ser Val Ser Val Leu Gly Asp Ile Leu Gly Ser Ala MetGln 945 950 955 960 Asn Thr Gln Asn Leu Leu Gln Met Pro Tyr Gly Cys GlyGlu Gln Asn 965 970 975 Met Val Leu Phe Ala Pro Asn Ile Tyr Val Leu AspTyr Leu Asn Glu 980 985 990 Thr Gln Gln Leu Thr Pro Glu Val Lys Ser LysAla Ile Gly Tyr Leu 995 1000 1005 Asn Thr Gly Tyr Gln Arg Gln Leu AsnTyr Lys His Tyr Asp Gly Ser 1010 1015 1020 Tyr Ser Thr Phe Gly Glu ArgTyr Gly Arg Asn Gln Gly Asn Thr Trp 1025 1030 1035 1040 Leu Thr Ala PheVal Leu Lys Thr Phe Ala Gln Ala Arg Ala Tyr Ile 1045 1050 1055 Phe IleAsp Glu Ala His Ile Thr Gln Ala Leu Ile Trp Leu Ser Gln 1060 1065 1070Arg Gln Lys Asp Asn Gly Cys Phe Arg Ser Ser Gly Ser Leu Leu Asn 10751080 1085 Asn Ala Ile Lys Gly Gly Val Glu Asp Glu Val Thr Leu Ser AlaTyr 1090 1095 1100 Ile Thr Ile Ala Leu Leu Glu Ile Pro Leu Thr Val ThrHis Pro Val 1105 1110 1115 1120 Val Arg Asn Ala Leu Phe Cys Leu Glu SerAla Trp Lys Thr Ala Gln 1125 1130 1135 Glu Gly Asp His Gly Ser His ValTyr Thr Lys Ala Leu Leu Ala Tyr 1140 1145 1150 Ala Phe Ala Leu Ala GlyAsn Gln Asp Lys Arg Lys Glu Val Leu Lys 1155 1160 1165 Ser Leu Asn GluGlu Ala Val Lys Lys Asp Asn Ser Val His Trp Glu 1170 1175 1180 Arg ProGln Lys Pro Lys Ala Pro Val Gly His Phe Tyr Glu Pro Gln 1185 1190 11951200 Ala Pro Ser Ala Glu Val Glu Met Thr Ser Tyr Val Leu Leu Ala Tyr1205 1210 1215 Leu Thr Ala Gln Pro Ala Pro Thr Ser Glu Asp Leu Thr SerAla Thr 1220 1225 1230 Asn Ile Val Lys Trp Ile Thr Lys Gln Gln Asn AlaGln Gly Gly Phe 1235 1240 1245 Ser Ser Thr Gln Asp Thr Val Val Ala LeuHis Ala Leu Ser Lys Tyr 1250 1255 1260 Gly Ala Ala Thr Phe Thr Arg ThrGly Lys Ala Ala Gln Val Thr Ile 1265 1270 1275 1280 Gln Ser Ser Gly ThrPhe Ser Ser Lys Phe Gln Val Asp Asn Asn Asn 1285 1290 1295 Arg Leu LeuLeu Gln Gln Val Ser Leu Pro Glu Leu Pro Gly Glu Tyr 1300 1305 1310 SerMet Lys Val Thr Gly Glu Gly Cys Val Tyr Leu Gln Thr Ser Leu 1315 13201325 Lys Tyr Asn Ile Leu Pro Glu Lys Glu Glu Phe Pro Phe Ala Leu Gly1330 1335 1340 Val Gln Thr Leu Pro Gln Thr Cys Asp Glu Pro Lys Ala HisThr Ser 1345 1350 1355 1360 Phe Gln Ile Ser Leu Ser Val Ser Tyr Thr GlySer Arg Ser Ala Ser 1365 1370 1375 Asn Met Ala Ile Val Asp Val Lys MetVal Ser Gly Phe Ile Pro Leu 1380 1385 1390 Lys Pro Thr Val Lys Met LeuGlu Arg Ser Asn His Val Ser Arg Thr 1395 1400 1405 Glu Val Ser Ser AsnHis Val Leu Ile Tyr Leu Asp Lys Val Ser Asn 1410 1415 1420 Gln Thr LeuSer Leu Phe Phe Thr Val Leu Gln Asp Val Pro Val Arg 1425 1430 1435 1440Asp Leu Lys Pro Ala Ile Val Lys Val Tyr Asp Tyr Tyr Glu Thr Asp 14451450 1455 Glu Phe Ala Ile Ala Glu Tyr Asn Ala Pro Cys Ser Lys Asp LeuGly 1460 1465 1470 Asn Ala 14 75 PRT Homo sapiens 14 Asp Met Gly Leu LysAla Phe Thr Asn Ser Lys Ile Arg Lys Pro Lys 1 5 10 15 Met Cys Pro GlnLeu Gln Gln Tyr Glu Met His Gly Pro Glu Gly Leu 20 25 30 Arg Val Gly PheTyr Glu Ser Asp Val Met Gly Arg Gly His Ala Arg 35 40 45 Leu Val His ValGlu Glu Pro His Thr Glu Thr Val Arg Lys Tyr Phe 50 55 60 Pro Glu Thr TrpIle Trp Asp Leu Val Val Val 65 70 75 15 1474 PRT Homo sapiens 15 Met GlyLys Asn Lys Leu Leu His Pro Ser Leu Val Leu Leu Leu Leu 1 5 10 15 ValLeu Leu Pro Thr Asp Ala Ser Val Ser Gly Lys Pro Gln Tyr Met 20 25 30 ValLeu Val Pro Ser Leu Leu His Thr Glu Thr Thr Glu Lys Gly Cys 35 40 45 ValLeu Leu Ser Tyr Leu Asn Glu Thr Val Thr Val Ser Ala Ser Leu 50 55 60 GluSer Val Arg Gly Asn Arg Ser Leu Phe Thr Asp Leu Glu Ala Glu 65 70 75 80Asn Asp Val Leu His Cys Val Ala Phe Ala Val Pro Lys Ser Ser Ser 85 90 95Asn Glu Glu Val Met Phe Leu Thr Val Gln Val Lys Gly Pro Thr Gln 100 105110 Glu Phe Lys Lys Arg Thr Thr Val Met Val Lys Asn Glu Asp Ser Leu 115120 125 Val Phe Val Gln Thr Asp Lys Ser Ile Tyr Lys Pro Gly Gln Thr Val130 135 140 Lys Phe Arg Val Val Ser Met Asp Glu Asn Phe His Pro Leu AsnGlu 145 150 155 160 Leu Ile Pro Leu Val Tyr Ile Gln Asp Pro Lys Gly AsnArg Ile Ala 165 170 175 Gln Trp Gln Ser Phe Gln Leu Glu Gly Gly Leu LysGln Phe Ser Phe 180 185 190 Pro Leu Ser Ser Glu Pro Phe Gln Gly Ser TyrLys Val Val Val Gln 195 200 205 Lys Lys Ser Gly Gly Arg Thr Glu His ProPhe Thr Val Glu Glu Phe 210 215 220 Val Leu Pro Lys Phe Glu Val Gln ValThr Val Pro Lys Ile Ile Thr 225 230 235 240 Ile Leu Glu Glu Glu Met AsnVal Ser Val Cys Gly Leu Tyr Thr Tyr 245 250 255 Gly Lys Pro Val Pro GlyHis Val Thr Val Ser Ile Cys Arg Lys Tyr 260 265 270 Ser Asp Ala Ser AspCys His Gly Glu Asp Ser Gln Ala Phe Cys Glu 275 280 285 Lys Phe Ser GlyGln Leu Asn Ser His Gly Cys Phe Tyr Gln Gln Val 290 295 300 Lys Thr LysVal Phe Gln Leu Lys Arg Lys Glu Tyr Glu Met Lys Leu 305 310 315 320 HisThr Glu Ala Gln Ile Gln Glu Glu Gly Thr Val Val Glu Leu Thr 325 330 335Gly Arg Gln Ser Ser Glu Ile Thr Arg Thr Ile Thr Lys Leu Ser Phe 340 345350 Val Lys Val Asp Ser His Phe Arg Gln Gly Ile Pro Phe Phe Gly Gln 355360 365 Val Arg Leu Val Asp Gly Lys Gly Val Pro Ile Pro Asn Lys Val Ile370 375 380 Phe Ile Arg Gly Asn Glu Ala Asn Tyr Tyr Ser Asn Ala Thr ThrAsp 385 390 395 400 Glu His Gly Leu Val Gln Phe Ser Ile Asn Thr Thr AsnVal Met Gly 405 410 415 Thr Ser Leu Thr Val Arg Val Asn Tyr Lys Asp ArgSer Pro Cys Tyr 420 425 430 Gly Tyr Gln Trp Val Ser Glu Glu His Glu GluAla His His Thr Ala 435 440 445 Tyr Leu Val Phe Ser Pro Ser Lys Ser PheVal His Leu Glu Pro Met 450 455 460 Ser His Glu Leu Pro Cys Gly His ThrGln Thr Val Gln Ala His Tyr 465 470 475 480 Ile Leu Asn Gly Gly Thr LeuLeu Gly Leu Lys Lys Leu Ser Phe Tyr 485 490 495 Tyr Leu Ile Met Ala LysGly Gly Ile Val Arg Thr Gly Thr His Gly 500 505 510 Leu Leu Val Lys GlnGlu Asp Met Lys Gly His Phe Ser Ile Ser Ile 515 520 525 Pro Val Lys SerAsp Ile Ala Pro Val Ala Arg Leu Leu Ile Tyr Ala 530 535 540 Val Leu ProThr Gly Asp Val Ile Gly Asp Ser Ala Lys Tyr Asp Val 545 550 555 560 GluAsn Cys Leu Ala Asn Lys Val Asp Leu Ser Phe Ser Pro Ser Gln 565 570 575Ser Leu Pro Ala Ser His Ala His Leu Arg Val Thr Ala Ala Pro Gln 580 585590 Ser Val Cys Ala Leu Arg Ala Val Asp Gln Ser Val Leu Leu Met Lys 595600 605 Pro Asp Ala Glu Leu Ser Ala Ser Ser Val Tyr Asn Leu Leu Pro Glu610 615 620 Lys Asp Leu Thr Gly Phe Pro Gly Pro Leu Asn Asp Gln Asp AsnGlu 625 630 635 640 Asp Cys Ile Asn Arg His Asn Val Tyr Ile Asn Gly IleThr Tyr Thr 645 650 655 Pro Val Ser Ser Thr Asn Glu Lys Asp Met Tyr SerPhe Leu Glu Asp 660 665 670 Met Gly Leu Lys Ala Phe Thr Asn Ser Lys IleArg Lys Pro Lys Met 675 680 685 Cys Pro Gln Leu Gln Gln Tyr Glu Met HisGly Pro Glu Gly Leu Arg 690 695 700 Val Gly Phe Tyr Glu Ser Asp Val MetGly Arg Gly His Ala Arg Leu 705 710 715 720 Val His Val Glu Glu Pro HisThr Glu Thr Val Arg Lys Tyr Phe Pro 725 730 735 Glu Thr Trp Ile Trp AspLeu Val Val Val Asn Ser Ala Gly Val Ala 740 745 750 Glu Val Gly Val ThrVal Pro Asp Thr Ile Thr Glu Trp Lys Ala Gly 755 760 765 Ala Phe Cys LeuSer Glu Asp Ala Gly Leu Gly Ile Ser Ser Thr Ala 770 775 780 Ser Leu ArgAla Phe Gln Pro Phe Phe Val Glu Leu Thr Met Pro Tyr 785 790 795 800 SerVal Ile Arg Gly Glu Ala Phe Thr Leu Lys Ala Thr Val Leu Asn 805 810 815Tyr Leu Pro Lys Cys Ile Arg Val Ser Val Gln Leu Glu Ala Ser Pro 820 825830 Ala Phe Leu Ala Val Pro Val Glu Lys Glu Gln Ala Pro His Cys Ile 835840 845 Cys Ala Asn Gly Arg Gln Thr Val Ser Trp Ala Val Thr Pro Lys Ser850 855 860 Leu Gly Asn Val Asn Phe Thr Val Ser Ala Glu Ala Leu Glu SerGln 865 870 875 880 Glu Leu Cys Gly Thr Glu Val Pro Ser Val Pro Glu HisGly Arg Lys 885 890 895 Asp Thr Val Ile Lys Pro Leu Leu Val Glu Pro GluGly Leu Glu Lys 900 905 910 Glu Thr Thr Phe Asn Ser Leu Leu Cys Pro SerGly Gly Glu Val Ser 915 920 925 Glu Glu Leu Ser Leu Lys Leu Pro Pro AsnVal Val Glu Glu Ser Ala 930 935 940 Arg Ala Ser Val Ser Val Leu Gly AspIle Leu Gly Ser Ala Met Gln 945 950 955 960 Asn Thr Gln Asn Leu Leu GlnMet Pro Tyr Gly Cys Gly Glu Gln Asn 965 970 975 Met Val Leu Phe Ala ProAsn Ile Tyr Val Leu Asp Tyr Leu Asn Glu 980 985 990 Thr Gln Gln Leu ThrPro Glu Ile Lys Ser Lys Ala Ile Gly Tyr Leu 995 1000 1005 Asn Thr GlyTyr Gln Arg Gln Leu Asn Tyr Lys His Tyr Asp Gly Ser 1010 1015 1020 TyrSer Thr Phe Gly Glu Arg Tyr Gly Arg Asn Gln Gly Asn Thr Trp 1025 10301035 1040 Leu Thr Ala Phe Val Leu Lys Thr Phe Ala Gln Ala Arg Ala TyrIle 1045 1050 1055 Phe Ile Asp Glu Ala His Ile Thr Gln Ala Leu Ile TrpLeu Ser Gln 1060 1065 1070 Arg Gln Lys Asp Asn Gly Cys Phe Arg Ser SerGly Ser Leu Leu Asn 1075 1080 1085 Asn Ala Ile Lys Gly Gly Val Glu AspGlu Val Thr Leu Ser Ala Tyr 1090 1095 1100 Ile Thr Ile Ala Leu Leu GluIle Pro Leu Thr Val Thr His Pro Val 1105 1110 1115 1120 Val Arg Asn AlaLeu Phe Cys Leu Glu Ser Ala Trp Lys Thr Ala Gln 1125 1130 1135 Glu GlyAsp His Gly Ser His Val Tyr Thr Lys Ala Leu Leu Ala Tyr 1140 1145 1150Ala Phe Ala Leu Ala Gly Asn Gln Asp Lys Arg Lys Glu Val Leu Lys 11551160 1165 Ser Leu Asn Glu Glu Ala Val Lys Lys Asp Asn Ser Val His TrpGlu 1170 1175 1180 Arg Pro Gln Lys Pro Lys Ala Pro Val Gly His Phe TyrGlu Pro Gln 1185 1190 1195 1200 Ala Pro Ser Ala Glu Val Glu Met Thr SerTyr Val Leu Leu Ala Tyr 1205 1210 1215 Leu Thr Ala Gln Pro Ala Pro ThrSer Glu Asp Leu Thr Ser Ala Thr 1220 1225 1230 Asn Ile Val Lys Trp IleThr Lys Gln Gln Asn Ala Gln Gly Gly Phe 1235 1240 1245 Ser Ser Thr GlnAsp Thr Val Val Ala Leu His Ala Leu Ser Lys Tyr 1250 1255 1260 Gly AlaAla Thr Phe Thr Arg Thr Gly Lys Ala Ala Gln Val Thr Ile 1265 1270 12751280 Gln Ser Ser Gly Thr Phe Ser Ser Lys Phe Gln Val Asp Asn Asn Asn1285 1290 1295 Arg Leu Leu Leu Gln Gln Val Ser Leu Pro Glu Leu Pro GlyGlu Tyr 1300 1305 1310 Ser Met Lys Val Thr Gly Glu Gly Cys Val Tyr LeuGln Thr Ser Leu 1315 1320 1325 Lys Tyr Asn Ile Leu Pro Glu Lys Glu GluPhe Pro Phe Ala Leu Gly 1330 1335 1340 Val Gln Thr Leu Pro Gln Thr CysAsp Glu Pro Lys Ala His Thr Ser 1345 1350 1355 1360 Phe Gln Ile Ser LeuSer Val Ser Tyr Thr Gly Ser Arg Ser Ala Ser 1365 1370 1375 Asn Met AlaIle Val Asp Val Lys Met Val Ser Gly Phe Ile Pro Leu 1380 1385 1390 LysPro Thr Val Lys Met Leu Glu Arg Ser Asn His Val Ser Arg Thr 1395 14001405 Glu Val Ser Ser Asn His Val Leu Ile Tyr Leu Asp Lys Val Ser Asn1410 1415 1420 Gln Thr Leu Ser Leu Phe Phe Thr Val Leu Gln Asp Val ProVal Arg 1425 1430 1435 1440 Asp Leu Lys Pro Ala Ile Val Lys Val Tyr AspTyr Tyr Glu Thr Asp 1445 1450 1455 Glu Phe Ala Ile Ala Glu Tyr Asn AlaPro Cys Ser Lys Asp Leu Gly 1460 1465 1470 Asn Ala

What is claimed is:
 1. A method for identifying a polymorphism orcombination of polymorphisms associated with an A2M-mediated disease ordisorder, comprising testing one or more polymorphisms in an A2M geneindividually and/or in combinations for genetic association with anA2M-mediated disease or disorder, wherein the one or more polymorphismsis/are selected from the group consisting of 6i, 12i.1, 12i.2, 12e, 14e,14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e.
 2. A method foridentifying a polymorphism or combination of polymorphisms associatedwith a neurodegenerative disease or disorder, comprising testing one ormore polymorphisms in an A2M gene individually and/or in combinationsfor genetic association with a neurodegenerative disease or disorder,wherein the one or more polymorphisms is/are selected from the groupconsisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i,21i, 28i and 30e.
 3. The method of claim 1, wherein the nucleotide at 6iis A, the nucleotide at 12i.1 is G, the nucleotide at 12i.2 is T, thenucleotide at 12e is T, the nucleotide at 14e is C, the nucleotide at14i.2 is C, the nucleotide at 17i.1 is G, the nucleotide at 20e is T,the nucleotide at 20i is G, the nucleotide at 21i is C, the nucleotideat 28i is T and the nucleotide at 30e is C, or the complementartnucleotide thereof.
 4. The method of claim 2, wherein the nucleotide at6i is A, the nucleotide at 12i.1 is G, the nucleotide at 12i.2 is T, thenucleotide at 12e is T, the nucleotide at 14e is C, the nucleotide at14i.2 is C, the nucleotide at 17i.1 is G, the nucleotide at 20e is T,the nucleotide at 20i is G, the nucleotide at 21i is C, the nucleotideat 28i is T and the nucleotide at 30e is C, or the complementartnucleotide thereof.
 5. The method of claim 2, wherein the disease isAlzheimer's disease.
 6. A method of genotyping a cell comprising:obtaining from an individual a biological sample containing analpha-2-macroglobulin nucleic acid or portion thereof, and determiningthe identity of one or more nucleotides in said alpha-2-macroglobulinnucleic acid or portion thereof wherein said one or more nucleotides arelocated at a position selected from the group consisting of 6i, 12i.1,12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e.
 7. Themethod of claim 6, wherein said alpha-2-macroglobulin nucleic acid isgenomic DNA.
 8. The method of claim 6, wherein saidalpha-2-macroglobulin nucleic acid is RNA.
 9. The method of claim 6,comprising determining the identity of one or more nucleotides at aposition selected from the group consisting of 6i, 12e, 14i.1 and 20e.10. The method of claim 9, further comprising determining the identityof one or more nucleotides at position 18i.
 11. The method of claim 6,comprising determining the identity of one or more nucleotides at aposition selected from the group consisting of 6i, 12e, 14i.1 and 21i.12. The method of claim 11, further comprising determining the identityof one or more nucleotides at position 18i.
 13. The method of claim 6,comprising determining the identity of one or more nucleotides at aposition selected from the group consisting of 12e, 14i.1 and 21 i. 14.The method of claim 13, further comprising determining the identity ofone or more nucleotides at a position selected from the group consistingof 18i and 24e.
 15. The method of claim 6, comprising determining theidentity of one or more nucleotides at a position selected from thegroup consisting of 14i. 1, 20e and 21 i.
 16. The method of claim 15,further comprising determining the identity of one or more nucleotidesat a position selected from the group consisting of 18i and 24e.
 17. Themethod of claim 6, comprising determining the identity of one or morenucleotides at a position selected from the group consisting of 20e, 21i and 28e.
 18. The method of claim 17, further comprising determiningthe identity of one or more nucleotides at a position selected from thegroup consisting of 18i and 24e.
 19. The method of claim 6, comprisingdetermining the identity of one or more nucleotides at a positionselected from the group consisting of 6i, 12e, 14i.1 and 21i.
 20. Themethod of claim 19, further comprising determining the identity of oneor more nucleotides at a position selected from the group consisting of18i and 24e.
 21. A method of genotyping a cell comprising: obtainingfrom an individual a biological sample containing analpha-2-macroglobulin polypeptide or portion thereof; and determiningthe identity of one or more amino acids in said alpha-2-macroglobulinpolypeptide or portion thereof wherein said one or more amino acids arelocated at a position selected from the group consisting of 14e, 20e and30e.
 22. A method of identifying a subject at risk for Alzheimer'sDisease, said method comprising: obtaining from said subject abiological sample containing an alpha-2-macroglobulin nucleic acid orportion thereof; and determining the presence or absence of one or morepolymorphisms or mutations in said alpha-2-macroglobulin nucleic acid orportion thereof wherein said one or more polymorphisms or mutationsoccur at a position selected from the group consisting of 6i, 12i.1,12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1, 20e, 20i, 21i, 28i and 30e. 23.The method of claim 22, wherein said alpha-2-macroglobulin nucleic acidis genomic DNA.
 24. The method of claim 22, wherein saidalpha-2-macroglobulin nucleic acid is RNA.
 25. The method of claim 22,wherein the nucleotide at 6i is A, the nucleotide at 12i.1 is G, thenucleotide at 12i.2 is T, the nucleotide at 12e is T, the nucleotide at14e is C, the nucleotide at 14i.2 is C, the nucleotide at 17i.1 is G,the nucleotide at 20e is T, the nucleotide at 20i is G, the nucleotideat 21i is C, the nucleotide at 28i is T and the nucleotide at 30e is Cor the complemtary nucleotides thereof.
 26. The method of claim 22,comprising determining the presence or absence of one or morepolymorphisms at a position selected from the group consisting of 6i,12e, 14i.1 and 20e.
 27. The method of claim 26, further comprisingdetermining the presence or absence of one or more polymorphisms atposition 18i.
 28. The method of claim 22, comprising determining thepresence or absence of one or more polymorphisms at a position selectedfrom the group consisting of 6i, 12e, 14i.1 and 21i.
 29. The method ofclaim 28, further comprising determining the presence or absence of oneor more polymorphisms at position 18i.
 30. The method of claim 22,comprising determining the presence or absence of one or morepolymorphisms at a position selected from the group consisting of 12e,14i.1 and 21i.
 31. The method of claim 30, further comprisingdetermining the presence or absence of one or more polymorphisms at aposition selected from the group consisting of 18i and 24e.
 32. Themethod of claim 22, comprising determining the presence or absence ofone or more polymorphisms at a position selected from the groupconsisting of 14i.1, 20e and 21i.
 33. The method of claim 32, furthercomprising determining the presence or absence of one or morepolymorphisms at a position selected from the group consisting of 18iand 24e.
 34. The method of claim 22, comprising determining the presenceor absence of one or more polymorphisms at a position selected from thegroup consisting of 20e, 21i and 28e.
 35. The method of claim 34,further comprising determining the presence or absence of one or morepolymorphisms at a position selected from the group consisting of 18iand 24e.
 36. The method of claim 22, comprising determining the presenceor absence of one or more polymorphisms at a position selected from thegroup consisting of 6i, 12e, 14i.1 and 21i.
 37. The method of claim 36,further comprising determining the presence or absence of one or morepolymorphisms at a position selected from the group consisting of 18iand 24e
 38. The method of claim 22, comprising determining the presenceor absence of one or more polymorphisms at a position selected from thegroup consisting of 12e, 12i and 28i.
 39. The method of claim 38,wherein the nucleotide at position 12e is T, or the complement thereof,the nucleotide at position 21i is A, or the complement thereof and thenucleotide at position 28i is A, or the complement thereof.
 40. A methodof identifying a subject at risk for Alzheimer's Disease, said methodcomprising: obtaining from said subject a biological sample containingan alpha-2-macroglobulin polypeptide or portion thereof; and determiningthe presence or absence of one or more polymorphisms or mutations insaid alpha-2-macroglobulin polypeptide or portion thereof wherein saidone or more polymorphisms or mutations occur at a position selected fromthe group consisting of 14e, 20e and 30e.
 41. A method of identifying acompound that modulates an alpha-2-macroglobulin activity comprising:providing a plurality of cells that express the LRP receptor; contactingsaid cells with a candidate compound; contacting said cells with analpha-2-macroglobulin polypeptide comprising at least one polymorphismor mutation having a position selected from the group consisting of 14e,20e, and 30e; and identifying a compound that modulates analpha-2-macroglobulin activity.
 42. The method of claim 41, wherein saidalpha-2-macroglobulin activity is an interaction of saidalpha-2-macroglobulin polypeptide with the LRP receptor.
 43. The methodof claim 41, wherein said alpha-2-macroglobulin activity is thedegradation of said alpha-2-macroglobulin polypeptide.
 44. The method ofclaim 41, wherein said alpha-2-macroglobulin activity is a proteaseinhibitor activity.
 45. The method of claim 41, wherein saidalpha-2-macroglobulin activity is the clearance of saidalpha-2-macroglobulin polypeptide.
 46. The method of claim 41, whereinsaid cells are contacted with an alpha-2-macroglobulin polypeptide inthe presence of amyloid β.
 47. The method of claim 46, wherein saidalpha-2-macroglobulin activity is an interaction of amyloid β or saidalpha-2-macroglobulin polypeptide with the LRP receptor.
 48. The methodof claim 47, wherein said alpha-2-macroglobutin mediates clearance ofamyloid β.
 49. A method of identifying a compound that modulates analpha-2-macroglobulin activity comprising: providing analpha-2-macroglobulin polypeptide comprising at least one of thepolymorphisms or mutations having a position selected from the groupconsisting of 14e, 20e, and 30e; contacting said alpha-2-macroglobulinpolypeptide with said compound; contacting said alpha-2-macroglobulinpolypeptide with methylamine; and identifying a compound that modulatesan alpha-2-macroglobulin activity by detecting a modulation in theactivation of said alpha-2-macroglobulin polypeptide.
 50. A method ofidentifying a compound that modulates an alpha-2-macroglobulin activitycomprising: providing an alpha-2-macroglobulin polypeptide comprising atleast one of the polymorphisms or mutations having a position selectedfrom the group consisting of 14e, 20e, and 30e; contacting saidalpha-2macroglobulin polypeptide with said compound; contacting saidalpha-2-macroglobulin polypeptide with amyloid β; and identifying acompound that modulates an alpha-2-macroglobulin activity by detecting amodulation in the formation of a complex of amyloid β and saidalpha-2-macroglobulin polypeptide.
 51. A method of making apharmaceutical comprising: identifying a compound by a method of claim41; and incorporating said compound into a pharmaceutical.
 52. Apurified or isolated nucleic acid comprising an alpha-2-macroglobulinsequence having a polymorphism or mutation at a position selected fromthe group consisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i.2, 17i.1,20e, 20i, 21i, 28i and 30e, wherein the nucleotide or nucleotidesequence at said position is other than an A2M-1.
 53. The purified orisolated nucleic acid of claim 52, wherein said alpha-2-macroglobulinsequence is SEQ ID NO: 1 or a sequence complementary thereto.
 54. Thepurified or isolated nucleic acid of claim 53, wherein the nucleotide ornucleotide sequence at said position is A2M-2.
 55. The purified orisolated nucleic acid of claim 52, wherein said alpha-2-macroglobulinsequence is selected from the group consisting of SEQ ID NOs: 2-8 andsaid polymorphism of mutation is at a position selected from the groupconsisting of 14e, 20e and 30e.
 56. The purified or isolated nucleicacid of claim 55, wherein the nucleotide or nucleotide sequence at saidposition is A2M-2.
 57. A purified or isolated nucleic acid comprising afragment of at least 16 consecutive nucleotides of SEQ ID NO: 1 having apolymorphism or mutation at a position selected from the groupconsisting of 6i, 12i.1, 12i.2, 12e, 14e, 14i.1, 14i, 17i.1, 20e, 20i,21i, 28i and 30e, wherein the nucleotide or nucleotide at said positionis other than an A2M-1 or a sequence complementary thereto.
 58. Thepurified or isolated nucleic acid of claim 56, wherein the nucleotide ornucleotide sequence at said position is A2M-2.
 59. A purified orisolated polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 9-15 having a polymorphism or mutationat a position selected from the group consisting of 14e, 20e and 30e,wherein the amino acid at said position is other than A2M-1.
 60. Thepurified or isolated polypeptide of claim 59, wherein the amino acid atsaid position is A2M-2.
 61. A purified or isolated polypeptidecomprising a fragment of an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 9-15 having a polymorphism or mutation at aposition selected from the group consisting of 14e, 20e and 30e, whereinthe amino acid mutation at said position is other than A2M-1.
 62. Thepurified or isolated polypeptide of claim 61, wherein the amino acid atsaid position is A2M-2.
 63. A recombinant vector comprising the nucleicacid claim
 57. 64. A cultured cell comprising the nucleic acid of claim57.
 65. A cultured cell comprising the polypeptide of claim
 61. 66. Acultured cell comprising the recombinant vector of claim
 63. 67. Anisolated or purified antibody that specifically binds to the polypeptideof claim
 61. 68. The antibody of claim 67, wherein said antibody ismonoclonal.
 69. A method of expressing an alpha-2-macroglobulinpolypeptide comprising: providing a construct comprising a promoteroperably linked to an alpha-2-macroglobulin nucleic acid having apolymorphism or mutation at a position selected from the groupconsisting of 14e, 20e and 30e, wherein the nucleotide at said positionis other than an A2M-1; and expressing said alpha-2-macroglobulin fromsaid construct.
 70. The method of claim 69, wherein said nucleotide atsaid position is A2M-2.